CN1748031A - Bacteria and process for producing chemical compounds by said bacteria - Google Patents

Bacteria and process for producing chemical compounds by said bacteria Download PDF

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CN1748031A
CN1748031A CNA2004800035293A CN200480003529A CN1748031A CN 1748031 A CN1748031 A CN 1748031A CN A2004800035293 A CNA2004800035293 A CN A2004800035293A CN 200480003529 A CN200480003529 A CN 200480003529A CN 1748031 A CN1748031 A CN 1748031A
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gene
orf
lysc
allelotrope
coryneform bacterium
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B·巴特
C·赖嫩
B·黑德里希
G·蒂尔巴赫
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Evonik Operations GmbH
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Abstract

The invention relates to coryneform bacteria which have, in addition to at least one copy, present at the natural site (locus), of an open reading frame (ORF), gene or allele which codes for the synthesis of a protein or an RNA, in each case a second, optionally third or fourth copy of this open reading frame (ORF), gene or allele at in each case a second, optionally third or fourth site in a form integrated into the chromosome and processes for the preparation of chemical compounds by fermentation of these bacteria.

Description

Bacterium and with the method for described bacterium production compound
Prior art
Compound is meant L-amino acid, VITAMIN, nucleosides and Nucleotide and D-amino acid especially, is used in human medicine, pharmaceutical industries, makeup, foodstuffs industry and the animal nutrition.
This many compounds is to prepare by fermentation coryneform bacterial strains, especially Corynebacterium glutamicum (Corynebacterium glutamicum).Because these materials are extremely important, therefore constantly make great efforts to improve these preparation methods.Improvement to this method can relate to the fermentation measure, the for example supply of stirring and oxygen, or the composition of the nutritional medium sugared concentration in the fermenting process for example, or form the product form gradually, perhaps the intrinsic output characteristic of microorganism self by for example ion exchange chromatography.
Utilized mutagenesis, selection and mutant system of selection to improve the output characteristic of these microorganisms.Obtained to produce specific compound in this way, the bacterial strain of resistance has been arranged, or for important metabolite in regulation and control be auxotrophic bacterial strain for metabolic antagonist.
Also utilized the recombinant DNA technology method for many years,, improved excellent bacillus (Corynebacterium) bacterial strain by single biosynthesis gene and the investigation production effect of increasing.
A kind of common method comprises by episomal replication plasmid some biosynthesis gene that increases in specified microorganisms.This method has a shortcoming to be, plasmid is lost (segregational instability) naturally in fermenting process (fermentation is usually directed to many generations in commercial run).
Another kind method comprises by the plasmid that does not duplicate in specified microorganisms duplicates specific biosynthesis gene.In this method, comprised that the plasmid of clone's biosynthesis gene is integrated into the karyomit(e) biosynthesis gene of this microorganism (people such as Reinscheid, Appliedand Environmental Microbiology 60 (1), 126-132 (1994); People such as Jetten, Applied Microbiology and Biotechnology 43 (1): 76-82 (1995)).A shortcoming of this method is, the nucleotide sequence of plasmid and select the nucleotide sequence of necessary antibiotics resistance gene to be retained in the microorganism.This processing and utilization for biological example matter is disadvantageous.In addition, the expert estimates, the desintegration owing in corresponding generation number (using always in such as industrial fermentation), pass through " Campbell's type exchange (Campbell type crossover) ", and such bacterial strain is unsettled.
Goal of the invention
The inventor's purpose is to provide new innovative approach for utilizing coryneform bacterium fermentative preparation compound.
Summary of the invention
The invention provides the coryneform bacterium of production compound, it is except synthetic synthetic protein of described compound or the open reading-frame (ORF) (ORF) of RNA of being used for of one or more coding that contains a copy at natural site (seat), outside gene or the allelotrope, also in one or more additional site, preferably at second, and contain one or more the 3rd or the 4th site to be integrated into chromosomal form alternatively, especially second, the described open reading-frame (ORF) (ORF) of the 3rd or the 4th copy alternatively, gene or allelotrope, wherein at least one described site is selected from intergenic region, prophage, defective phage or phage assembly or the described prophage of encoding, the DNA of defective phage or phage assembly.
Second, alternatively be not present in the 3rd or the 4th site in the microorganism can/make it possible to carry out the nucleotide sequence of episomal replication or swivel base, there is not the nucleotide sequence of giving its antibiotics resistance, second, the 3rd or the 4th site do not relate to for the growth of bacterium and essential open reading-frame (ORF) (ORF), gene or the allelotrope of generation of required compound alternatively.
The present invention also provides the method for one or more compounds of preparation, wherein takes following steps:
A) fermentation coryneform bacterium, this coryneform bacterium is used for open reading-frame (ORF) (ORF), gene or the allelotrope of the protein of synthetic described compound or RNA except there is one or more coding at natural site (seat),
B) also in some site with one or more, especially second, described open reading-frame (ORF) (ORF), gene or the allelotrope of the 3rd or the 4th copy are integrated into karyomit(e) alternatively, thus
C) at least one described site is selected from the DNA of intergenic region, prophage, defective phage or phage assembly or the described prophage of encoding, defective phage or phage assembly,
D) be not present in described site in the microorganism can/make it possible to carry out the nucleotide sequence of episomal replication or swivel base and do not have the nucleotide sequence of giving its antibiotics resistance,
E) under the condition that described open reading-frame (ORF) (ORF), gene or allelotrope can be expressed, carried out expression, the compound in concentrated broth body substratum and/or the bacterial cell,
F) separate this compound, alternatively
G) together with from the composition in fermentation broth and/or the biomass, until＞(greater than) 0 to 100wt%.
Activity in the intracellular reactive of respective egg white matter or concentration and the initial bacterium (parent bacterium or wild-type) or concentration are compared and are improved, when especially the nucleotide sequence of code for said proteins is crossed expression.General this raising is at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, is 1000% or 2000% to the maximum.
The present invention also provides so excellent bacilli-cell, be that it contains and is present in open reading-frame (ORF) (ORF), gene or allelotrope that natural site and coding be used for the protein of synthetic described compound (especially amino acid) or RNA preferably with described open reading-frame (ORF) (ORF), gene or the allelotrope of the 3rd or the 4th copy of arranged in series (tandem arrangement) direct neighbor, described in WO 03/014330.
" arranged in series " means, and two or more ORF, gene or allelotrope file are directly adjacent to each other and the identical arrangement mode of direction.The ORF of two or more direct neighbor, gene or allelic another kind of direction type can be called oppositely.
These coryneform bacteriums comprise described " arranged in series " for the copy in the site that is selected from intergenic region, prophage, defective phage or phage assembly in addition.
The present invention also provides the method for the described coryneform bacterium of described fermentation.
Detailed Description Of The Invention
Compound refers in particular to amino acid, VITAMIN, nucleosides and Nucleotide.The biosynthetic pathway of these compounds is known and available in the prior art.
Amino acid preferably means L-amino acid, the L-amino acid of protein source particularly, be selected from L-aspartic acid, altheine, L-Threonine, L-Serine, L-L-glutamic acid, L-glutamine, glycine, L-L-Ala, L-halfcystine, L-Xie Ansuan, L-methionine(Met), L-Isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-Histidine, L-Methionin, L-tryptophane, L-proline(Pro) and L-arginine and salt thereof, especially L-Methionin, L-methionine(Met) and L-Threonine.L-Methionin is very preferred.
The amino acid of protein source means the amino acid that appears in the natural protein, that is to say in the protein that appears at microorganism, plant, animal and human.
VITAMIN means, especially VITMAIN B1 (VitB1), Wei ShengsuB2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxol), vitamin B12 (cyanocobalamin), nicotinic acid/niacinamide, vitamin(e) M (folic acid) and vitamin-E (tocopherol) and salt thereof, pantothenic acid is preferred.
Nucleosides and Nucleotide mean, except that other, and S-adenosine-methionine(Met), inosine-5 '-single phosphoric acid and guanosine-5 '-single phosphoric acid and salt thereof.
Coryneform bacterium is the bacterium of Corynebacterium (Corynebacterium) particularly.In Corynebacterium, Corynebacterium glutamicum (Corynebacterium glutamicum), product ammonia rod bacillus (Corynebacterium ammoniagenes) and Corynebacteriumthermoaminogenes are preferred.Except that other, can be at K_mpfer and Kroppenstedt (Canadian Journal of Microbiology 42,989-1005 (1996)) and at US-A-5, find the taxonomic information of bacterial strain in this group bacterium in 250,434.
The suitable bacterial strain (especially) of Corynebacterium is known wild type strain
Corynebacterium glutamicum ATCC13032
Vinegar paddy rod bacillus (Corynebacterium acetoglutamicum) ATCC15806
Corynebacterium acctoacidophlum (Corynebacterium acetoacidophilum) ATCC13870
Lily hedysarum scoparium bacillus (Corynebacterium lilium) ATCC15990
Corynebacterium melassecola ATCC17965
Man of great strength's rod bacillus (Corynebacterium herculis) ATCC13868
Arthrobacter (Arthrobacter sp.) ATCC243
Brevibacterium chang-fua ATCC14017
Brevibacterium flavum (Brevibacterium flavum) ATCC14067
Brevibacterium (Brevibacterium lactofermentum) ATCC13869
Extension brevibacterium (Brevibacterium divaricatum) ATCC14020
Taibei tyrothricin (Brevibacterium taipei) ATCC13744 and
Have a liking for ammonia microbacterium (Microbacterium ammoniaphilum) ATCC21645 and mutant or bacterial strain, known such as prior art, derive and the mutant or the bacterial strain that come from the bacterial strain that produces compound.
The suitable bacterial strain (especially) that produces in the ammonia rod bacillus species is known wild type strain
Brevibacterium ammoniagenes (Brevibacterium ammoniagenes) ATCC6871
Brevibacterium ammoniagenes ATCC15137 and
Rod bacillus ATCC21084
With mutant or bacterial strain, known such as prior art, derive and the mutant or the bacterial strain that come from the bacterial strain that produces compound.
Suitable bacterial strain (especially) in the Corynebacterium thermoaminogenes species is known wild type strain
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium thermoaminogenes FERM BP-1540
Corynebacterium thermoaminogenes FERM BP-1541 and
Corynebacterium thermoaminogenes FERM BP-1542 and mutant or bacterial strain, known such as prior art, derive and the mutant or the bacterial strain that come from the bacterial strain that produces compound.
Have " ATCC " mark bacterial strain can available from American Type Culture Collection (AmericanType Culture Collection) (Manassas, VA, USA).The bacterial strain that has " FERM " mark can be available from national advanced industrial science and technology (the AIST TsukubaCentral 6 of institute (National Institute ofAdvanced Industrial Science and Technology), 1-1-1Higashi, Tsukuba Ibaraki, Japan).The bacterial strain of the Corynebacterium thermoaminogenes that is mentioned (FERM BP-1539, FERMBP-1540, FERM BP-1541 and FERM BP-1542) is described in US-A 5,250, in 434.
Open reading-frame (ORF) (ORF) is described one section coding or can coded protein or the nucleotide sequence of polypeptide or Yeast Nucleic Acid, can't point out the function of this section nucleotide sequence according to prior art.
After pointing out function, usually it is called gene for this nucleotide sequence fragment.
Allelotrope is generally understood as the alternative form that means a given gene.The difference of these forms is nucleotide sequence.
In the context of the invention, preferably use endogenous (also being species specificity) open reading-frame (ORF), gene or allelotrope.They mean and are present in a certain species colony, for example open reading-frame (ORF) in the Corynebacterium glutamicum, gene or allelotrope or its nucleotide sequence.
" open reading-frame (ORF) that is positioned at natural site (seat) (ORF), gene or the allelotrope of a copy " in the context of the invention is interpreted as and means, and ORF or gene or allelotrope are with respect to biological or play adjacent ORF or gene or allelic position in the eozoan such as being present in corresponding wild type or corresponding parent.
Therefore, for example, coding is from the lysC gene or the lysC of " feedback " resistance E.C. 2.7.2.4. of Corynebacterium glutamicum FBRAllelic natural site is meant, a side has lysC site or lysC seat or the 1ysC gene locus that the gene of direct neighbor or open reading-frame (ORF) orfX and leuA, opposite side have the asd gene of direct neighbor.
" feedback " resistance E.C. 2.7.2.4. is interpreted as and means such E.C. 2.7.2.4.: compare with the wild-type form, it reduces (at least 5 to 10%, 10% to 15% or 10% to 20%) for the susceptibility of the inhibition of the mixture of the mixture of Methionin and Threonine or AEC (amino-ethyl halfcystine) and Threonine or Methionin itself or AEC itself.The bacterial strain that produces L-Methionin comprises E.C. 2.7.2.4. such " feedback " resistance or desensitization usually.
The chromosomal nucleotide sequence of Corynebacterium glutamicum is known, and can be at patent application EP-A-1108790 and the (EMBL of European Molecular Bioglogy Laboratory, Heidelberg, Germany and Cambridge UK) find among the accession number of nucleotide sequence database (AccessionNo.) AX114121.The accession number of OrfX, leuA gene and asd gene nucleotide series is AX120364 (orfX), AX123517 (leuA) and AX123519 (asd).
Can also utilize other database, for example National Library of Medicine (Bethesda, MD, NCBI USA) (National Centerfor BiotechnologyInformation, NCBI).
Also can use other database, such as NCBI (NCBI, Bethesda, MD, USA) database, or Switzerland information biology institute (SwissInstitute of Bioinformatics, Swissprot, Geneva, Switzerland) database, or protein information resource database (Protein Information ResourceDatabase, PIR, Washington, U.S.) database.
" one or more, particularly second, the 3rd or the 4th site alternatively " is interpreted as and means the site that is different from " natural site ".It is also referred to as " target site " or " target sequence " hereinafter.It can also be called " integration site " or " conversion site ".This (a bit) site or the nucleotide sequence that is present in corresponding site are preferably placed in the karyomit(e), and are not to be that growth and required compound production are necessary usually.
Preferably with the chromosomal intergenic region of Corynebacterium glutamicum be generally comprised within prophage in the karyomit(e) and the DNA zone of coding defective phage or phage assembly is a target site.Such site example is shown in table 12 and 13.
For preparing coryneform bacterium of the present invention, by for example PCR or utilize restriction enzyme and after the gel extracting, at least separate the dna fragmentation that obtains carrying purpose ORF, gene, allelotrope or operon, comprise alternatively and expressing and/or adjustment signal (for example promotor, attenuator, modulin binding site, ribosome bind site, attenuator and terminator).Dna fragmentation length changes with the natural length(of wool) of specific ORF, gene, allelotrope or operon.Generally, this length is between 0.25 to 10kb or 0.5 to 10kb.Afterwards for this (a bit) dna fragmentation 5 '-provide the nucleotide sequence of target site with 3 '-end.Then, preferably, these dna fragmentations are transformed into purpose coryneform bacterium by in excellent bacillus, not duplicating or the limited carrier that duplicates only taking place; Isolate those and be integrated with purpose ORF, gene or allelic bacterium at target site; The target site place be not retained in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/nucleotide sequence that makes it possible to the nucleotide sequence of swivel base and give its antibiotics resistance.Certainly this method is made amendment, for example, make and at first target site is inserted (clone advances) carrier, and insert purpose ORF, gene, allelotrope or operon at described target site place subsequently.
The also corresponding preparation method that the coryneform bacterium of producing one or more compound is provided of the present invention comprises
A) separating at least one purpose ORF, gene or allelic nucleotide sequence comprise alternatively and expressing and/or adjustment signal,
B) for ORF, gene or allelic 5 ' and 3 ' end the nucleotide sequence of target site is provided, wherein said site is selected from intergenic region, prophage, defective phage or phage assembly,
C) purpose ORF, gene or the allelic nucleotide sequence that preferably will have a target site nucleotide sequence is integrated into and do not duplicate in coryneform bacterium or only carry out in the limited carrier that duplicates,
D) with b) or nucleotide sequence c) or carrier shift in the coryneform bacterium and
E) be separated in target site and integrated coryneform bacterium according to a) nucleotide sequence, the target site place do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.
Also preferred, the target site place does not keep the residue of used carrier sequence or species allogeneic dna sequence DNA, such as restricted cleavage site or especially can or make it possible to the nucleotide sequence that duplicates or transcribe in the bacterium that transforms, and the target site place does not keep the nucleotide sequence of giving antibiotics resistance.Maximum 24 of the DNA that is positioned at ORF, gene or allelotrope upstream or the downstream integrated like this, preferred maximum 12, especially preferred maximum 6 Nucleotide are retained in target site alternatively.
With coding be used for the ORF, gene of the protein of production compound or RNA, allelic 5 '-and 3 '-length of the terminal target site nucleotide sequence that adheres to mutually is minimum to be at least 100 base pairs.Can use nucleotide sequence length similarly is the DNA of at least 200,300,400,500,600,800,1000,1200,1400,16000,18000 or 20000 base pairs.Usually maximum length is no more than 3000,5000 or 10000 base pairs.Preferred length is at least 300 and be 3000 to the maximum.
By method of the present invention, the coryneform bacterium of preparation compound or the productivity of fermentation process are being selected from concentration (formed compound, based on unit volume), output (formed compound, based on the carbon source that is consumed) and product one or more characteristic aspect of forming speed (formed compound is based on the time) improve at least 0.5-1.0% or at least 1.0 to 1.5% or 1.5-2.0% at least.
Relevant such as chromosomal DNA, plasmid DNA separation and the guidance of such conventional genetic engineering method such as restriction enzyme operation can in people such as Sambrook (Molecular Cloning-A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press), find.Guidance about the conversion of excellent bacillus and joint can be at people such as Thierbach (Applied Microbiology and Biotechnology 29 except that other, 356-362 (1988)), people (Journal of Bacteriology 172 such as Sch_fer, 1663-1666 (1990) and Gene 145,69-73 (1994)) and among Schwarzer and the P ü hler (Bio/Technology 9,84-87 (1991)) find.
Only carry out the limited carrier that duplicates and be interpreted as and mean, the plasmid vector that duplicates or do not duplicate as the function of the condition of cultivating host or carrier.Therefore, people such as Nakamura (US-A-6,303,383) have described a kind of responsive to temperature type plasmid of coryneform bacterium, can only carry out limited duplicating when it equals/be lower than 31 ℃ in temperature.
In addition, for separating and identifying coryneform bacterium of the present invention, can utilize microbiological technique commonly used such as being coated with flat board, selection and screening, and gene engineering commonly used is such as DNA hybridization, polymerase chain reaction (PCR) and dna sequencing, and biochemical method commonly used is such as enzyme activity determination.
The present invention further provides the coryneform bacterium of producing L-Methionin, especially the bacterium of Corynebacterium, it produces the proteinic open reading-frame (ORF) (ORF) of Methionin except one or more coding that contains at least one copy at natural site (seat), outside gene or the allelotrope, also at second, being incorporated into intrachromosomal site at the 3rd or the 4th alternatively contains one or more, especially second, the described open reading-frame (ORF) (ORF) of the 3rd or the 4th copy alternatively, gene or allelotrope, wherein at least one described site is selected from intergenic region, prophage, defective phage or phage assembly or the described prophage of encoding, the DNA of defective phage or phage assembly.
Described site do not have in microorganism can/make it possible to carry out episomal replication or can/nucleotide sequence that makes it possible to carry out swivel base or give its antibiotics resistance.
The present invention provides the method for preparing L-Methionin in addition, comprising:
A) ferment described coryneform bacterium, especially Corynebacterium glutamicum,
Under the condition that can allow described open reading-frame (ORF) (ORF), gene or allelotrope to express,
B) the L-Methionin in the concentrated broth body substratum,
C) from fermentation broth, separate L-Methionin, alternatively
D) together with from the composition in fermentation broth and/or the biomass, until＞(greater than) 0 to 100%.
Open reading-frame (ORF) (ORF), gene or the allelic copy of Methionin " produce " is interpreted as and means, and all can have raising Methionin and produce this effect, preferred endogenous open reading-frame (ORF), gene or allelotrope after enhancings/mistakes expression.Enhancing is interpreted as and means that the IC of specific gene product, protein or enzyme or activity are with respect to the improve of the activity or the concentration of protein in the initial bacterium or enzyme.
These comprise following open reading-frame (ORF), gene or allelotrope: accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC except that other FBR, lysE, msiK, opcA, oxyR, ppc, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T.Their are summarized and explain in table 1.
These comprise, the lysC of " feedback " resistance E.C. 2.7.2.4. of especially encoding FBRAllelotrope.Various lysC FBRAllelotrope has provided in table 2 summarizes and explanation.
Following lysC FBRAllelotrope is preferred: lysC A279T (L-Ala according to the 279th of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by Threonine), lysCA279V (L-Ala according to the 279th of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by Xie Ansuan), lysC S301F (Serine according to the 301st of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by phenylalanine), lysC T308I (Threonine according to the 308th of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by Isoleucine), lysC S301Y (Threonine according to the 308th of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by tyrosine), lysC G345D (glycine according to the 345th of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by aspartic acid), lysC R320G (arginine according to the 320th of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by glycine), lysC T311I (Threonine according to the 311st of the coded aspartokinase zymoprotein of SEQ IDNO:2 is replaced by Isoleucine), lysC S381F (Serine according to the 381st of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by phenylalanine).
LysC FBRAllelotrope lysC T311I (Threonine according to the 311st of the coded aspartokinase zymoprotein of SEQ ID NO:2 is replaced by Isoleucine) is particularly preferred, and its nucleotide sequence is shown in SEQ ID NO:3; The proteinic aminoacid sequence of coded E.C. 2.7.2.4. is shown in SEQ ID NO:4.
In the 3rd or the 4th site, can integrate open reading-frame (ORF) (ORF), gene or the allelic the 3rd or the 4th copy of described production Methionin.Except that other, following open reading-frame (ORF), gene or nucleotide sequence can be used for this: aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi, poxB and zwa2, especially gene aecD, gluA, gluB, gluC, gluD and pck.These have provided in table 3 summarizes and explanation.
The site of being mentioned not only comprises the open reading-frame (ORF) mentioned or the coding region of gene certainly, comprise that also being positioned at the upstream is responsible for the zone or the nucleotide sequence of expressing and regulating and control, such as the binding site and the attenuator of ribosome bind site, promotor, modulin binding site, regulation and control Yeast Nucleic Acid.These zones are usually located in coding region upstream 1-800,1-600,1-400,1-200,1-100 or 1-50 the Nucleotide scope.Equally, be positioned at the zone in downstream, in being also included within such as transcription terminator.These zones are usually located in coding region downstream 1-400,1-200,1-100,1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region, just not have the nucleotide sequence of encoding function.At last, be generally comprised within prophage in the karyomit(e) or defective phage or phage assembly and can be used for this.
Prophage is interpreted as and means the phage of duplicating and do not form infectious particle with host genome, especially its genome.Defective phage is interpreted as and means the prophage of having lost the ability that forms so-called infectious particle owing to various sudden changes, especially its genome.Defective phage is also referred to as cryptic prophage.Prophage and defective phage often occur with the form that is incorporated in its host chromosome.Further description is arranged in the prior art, for example at the textbook of writing by Edward A.Birge (Bacterial and BacteriophageGenetics, the third edition, Springer-Verlag, New York, the U.S., 1994) or the textbook (Bakterienviren, Gustav Fischer Verlag, the Jena that write by people such as S.Klaus, Germany, 1992) in.
Represent the example of Corynebacterium glutamicum chromosomal region of the DNA of intergenic region, prophage, defective phage or phage assembly or the described prophage of encoding, defective phage or phage assembly to be shown in table 12 and 13.The position in DNA zone is with reference to the Genome Atlas of the Corynebacterium glutamicum ATCC 13032 shown in EP-A-1108790 or European Molecular Bioglogy Laboratory (EMBL, Heidelberg, Germany and Cambridge, the Britain) database.
With regard to the intergenic region of table 12, it is evident that to those skilled in the art, because different mutually and handle between wild-type and the wild type strain, can use according to the present invention and contain that to have at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and do not have encoding function with the described nucleotide sequence of table 12 be gene or allelic zone according to the sudden change of being accepted.
With regard to the DNA zone of the coding prophage, defective phage or the phage assembly that are selected from table 13, it is evident that to those skilled in the art, since different mutually and handle between wild-type and the wild type strain according to the sudden change of being accepted, can use according to the present invention and contain the zone that has at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and coding prophage, defective phage or phage assembly with the described nucleotide sequence of table 13.
Table 1
Produce open reading-frame (ORF), gene and the allelotrope of Methionin
Title Coded enzyme or proteinic explanation Reference Accession number
accBC Acyl group-CoA carboxylase EC 6.3.4.14 (acyl group-CoA carboxylase) People such as J_ger, Archives of Microbiology (1996) 166:76-82 EP1108790; WO0100805 U35023 AX123524 AX066441
accDA Acetyl-CoA carboxylase EC 6.4.1.2 (acetyl-CoA carboxylase) EP1055725 EP1108790 WO0100805 AX121013 AX066443
cstA Carbon Starvation Protein A (the hungry albumin A of carbon) EP1108790 WO0100804 AX120811 AX066109
cysD Sulfate Adenylyltransferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) EP1108790 AX123177
cysE Serine Acetyltrahsferase EC 2.3.1.30 (serine acetyltransferase) EP1108790 WO0100843 AX122902 AX063961
cysH 3 '-Phospho adenyl Sulfate Reductase EC 1.8.99.4 (3'-phosphoadenosine-5'-phosphosulfate reductase enzyme) EP1108790 WO0100842 AX123178 AX066001
cysK Cysteine Synthase EC 4.2.99.8 (cysteine synthase) EP1108790 WO0100843 AX122901 AX063963
cysN Sulfate Adenylyltrahsferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) EP1108790 AX123176 AX127152
cysQ Transport Protein CysQ (translocator cysQ) EP1108790 WO0100805 AX127145 AX066423
dapA Dihydrodipicolinate Synthase EC 4.2.1.52 (dihydrodi pyridine formic acid synthase) The people such as Bonnassie; The people such as Nucleic Acids Research 18:6421 (1990) Pisabarro, Journal of Bacteriology 175:2743-2749 (1993) EP1108790 WO0100805 EP0435132 EP1067192 EP1067193 X53993 Z21502 AX123560 AX063773
dapB Dihydrodipicolinate Reductase EC 1.3.1.26 (dihydrodi pyridine formic acid reductase enzyme) The people such as EP1108790 WO0100843 EP1067192 EP1067193 Pisabarro, Journal of Bacteriology 175:2743-2749 (1993) JP1998215883 JP1997322774 JP1997070291 JP1995075578 AX127149 AX063753 AX137723 AX137602 X67737 Z21502 E16749 E14520 E12773 E08900
dapC N-Succinyl Aminoketopimelate EP1108790 AX127146
Transaminase EC 2.6.1.17 (N-succinyl-diaminopimelate trans aminase) WO0100843 EP1136559 AX064219
dapD Tetrahydrodipicolinate Succinylase EC 2.3.1.117 (tetrahydrochysene two pyridine carboxylic acid succinylation enzymes) People such as EP1108790 WO0100843 Wehrmann, Journal of Bacteriology 180:3159-3165 (1998) AX127146 AX063757 AJ004934
dapE N-Succinyl Diaminopimelate Desuccinylase EC 3.5.1.18 (N-succinyldiaminopimelate desuccinylase) People such as EP1108790 WO0100843 Wehrmann, Microbiology 140:3349-3356 (1994) AX127146 AX063749 X81379
dapF Diaminopimelate Epimerase EC 5.1.1.7 (diaminopimelic acid epimerase) EP1108790 WO0100843 EP1085094 AX127149 AX063719 AX137620
ddh Diaminopimelate Dehydrogenase EC 1.4.1.16 (diaminopimelate dehydrogenase) The people such as EP1108790 WO0100843 Ishino; The people such as Nucleic Acids Research 15:3917-3917 (1987) JP1997322774 JP1993284970 Kim, Journal of Microbiology and Biotechnology 5:250-256 (1995) AX127152 AX063759 Y00151 E14511 E05776 D87976
dps DNA Protection Protein (albumen of between hunger period, protecting) EP1108790 AX127153
eno Enolase EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) People such as EP1108790 WO0100844 EP1090998 Hermann, Electrophoresis 19:3217-3221 (1998) AX127146 AX064945 AX136862
gap Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) People such as EP1108790 WO0100844 Eikmanns, Journal of Bacteriology 174:6076-6086 AX127148 AX064941 X59403
77(2)：237-251 (1989)
pgk Phosphoglycerate Kinase EC 2.7.2.3 (phosphoglyceric kinase) EP1108790 WO0100844 Eikmanns，Journal 0f Bacteriology 174：6076-6086 (1992) AX121838 AX127148 AX064943 X59403
pknA Protein Kinase A (protein kinase A) EP1108790 AX120131 AX120085
pknB Protein Kinase B (protein kinase B) EP1108790 AX120130 AX120085
pknD Protein Kinase D (protein kinase D) EP1108790 AX127150 AX122469 AX122468
pknG Protein Kinase G (protein kinase G) EP1108790 AX127152 AX123109
ppsA Phosphoenol Pyruvate Synthase EC 2.7.9.2 (phosphoenolpyruvate synthase) EP1108790 AX127144 AX120700 AX122469
ptsH Phosphotransferase System Protein H EC 2.7.1.69 (phosphotransferase system component H) EP1108790 WO0100844 AX122210 AX127149 AX069154
ptsI Phosphotransferase System Enzyme I EC 2.7.3.9 (phosphotransferase system enzyme I) EP1108790 AX122206 AX127149
ptsM Glukose-specific Phosphotransferase System Enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) People such as Lee, FEMS Microbiology Letters 119 (1-2): 137-145 (1994) L18874
pyc Pyruvate Carboxylase EC 6.4.1.1 (pyruvate carboxylase) People such as WO9918228 Peters-Wendisch, Microbiology 144:915-927 (1998) A97276 Y09548
pyc P458S Pyruvate Carboxylase EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S EP1108790
sigC Sigma Factor C EC 2.7.7.6 (the outer function substituted type Sigma Factors C of kytoplasm) EP1108790 AX120368 AX120085
sigD RNA Polymerase Sigma Factor D EC 2.7.7.6 EP1108790 AX120753 AX127144
(RNA polymerase Sigma Factors)
sigE Sigma Factor E EC 2.7.7.6 (the outer function substituted type Sigma Factors E of kytoplasm) EP1108790 AX127146 AX121325
sigH Sigma Factor H EC 2.7.7.6 (Sigma Factors SigH) EP1108790 AX127145 AX120939
sigM Sigma Factor M EC 2.7.7.6 (Sigma Factors SigM) EP1108790 AX123500 AX127145
tal Transaldolase EC 2.2.1.2 (transaldolase) WO0104325 AX076272
thyA Thymidylate Synthase EC 2.1.1.45 (thymidylate synthase) EP1108790 AX121026 AX127145
tkt Transketolase EC 2.2.1.1 (transketolase) People such as Ikeda, NCBI AB023377
tpi Triose Phosphate Isomerase EC 5.3.1.1 (triose-phosphate isomerase) Eikmanns，Journal of Bacteriology 174：6076-6086 (1992) X59403
zwa1 Cell Growth Factor 1 (somatomedin 1) EP1111062 AX133781
zwf Glucose 6-phosphate 1-Dehydrogenase EC 1.1.1.49 (G-6-P 1-desaturase) EP1108790 WO0104325 AX127148 AX121827 AX076272
zwf A213T Glucose 6-phosphate 1-Dehydrogenase EC 1.1.1.49 (G-6-P 1-desaturase) amino acid exchange A213T EP1108790
Table 2
The lysC of encoder feedback resistance E.C. 2.7.2.4. FBRAllelotrope
The allelotrope title More information Reference Accession number
lysC FBR-E05108 JP 1993184366-A (sequence 1) E05108
lys CFB-E06825 lysC A279T JP 1994062866-A (sequence 1) E06825
lysC FBR-E06826 lysC A279T JP 1994062866-A (sequence 2) E06826
lysC FBR-E06827 JP 1994062866-A (sequence 3) E06827
lysC FBR-E08177 JP 1994261766-A (sequence 1) E08177
lysC FBR-E08178 lysC A279T JP 1994261766-A (sequence 2) E08178
lysC FBR-E08179 lysC A279V JP 1994261766-A (sequence 3) E08179
lysC FBR-E08180 lysC S301F JP 1994261766-A (sequence 4) E08180
lysC FBR-E08181 lysC T308I JP 1994261766-A (sequence 5) E08181
lysC FBR-E08182 JP 1994261766-A (sequence 6) E08182
lysC FBR-E12770 JP 1997070291-A (sequence 13) E12770
lysC FBR-E14514 JP 1997322774-A (sequence 9) E14514
lysC FBR-E16352 JP 1998165180-A (sequence 3) E16352
lysC FBR-E16745 JP 1998215883-A (sequence 3) E16745
lysC FBR-E16746 JP 1998215883-A (sequence 4) E16746
lysC FBR-I74588 US 5688671-A (sequence 1) I74588
lysC FBR-I74589 lysC A279T US 5688671-A (sequence 2) I74589
lysC FBR-I74590 US 5688671-A (sequence 7) I74590
lysC FBR-I74591 lysC A279T US 5688671-A (sequence 8) I74591
lysC FBR-I74592 US 5688671-A (sequence 9) I74592
lysC FBR-I74593 lysC A279T US 5688671-A (sequence 10) I74593
lysC FBR-I74594 US 5688671-A (sequence 11) I74594
lysC FBR-I74595 lysC A279T US 5688671-A (sequence 12) I74595
lysC FBR-I74596 US 5688671-A (sequence 13) I74596
lysC FBR-I74597 lysC A279T US 5688671-A (sequence 14) I74597
lysC FBR-X57226 lysC S301Y People such as EP0387527 Kalinowski, Molecular and General Genetics 224:317-324 (1990) X57226
lysC FBR-L16848 lysC G345D Follettie and Sinskey NCBI Nucleotide Database(1990) L16848
lysC FBR-L27125 lysC R320G lysC G345D People such as Jetten, Applied Microbiology Biotechnology 43:76-82 (1995) L27125
lysC FBR lysC T311I WO0063388 (sequence 17)
lysC FBR lysC S301F US3732144
lysC FBR lysC S381F
lysC FBR JP6261766 (sequence 1)
lysC FBR lysC A279T JP6261766 (sequence 2)
lysC FBR lysC A279V JP6261766 (sequence 3)
lysC FBR lysCS 301F JP6261766 (sequence 4)
lysC FBR lysC T308I JP6261766 (sequence 5)
Table 3
Be used to integrate open reading-frame (ORF), gene and the allelic target site of producing Methionin
The gene title Coded enzyme or proteinic explanation Reference Accession number
aecD Beta C-S Lyase EC 2.6.1.1 (β C-S lyase) People such as Rossol, Journal of Bacteriology 174 (9): 2968-77 (1992) M89931
ccpA1 Catabolite Control Protein (catabolite control albumin A 1) WO0100844 EP1108790 AX065267 AX127147
ccpA2 Catabolite Control Protein (catabolite control albumin A 2) WO0100844 EP1108790 AX065267 AX121594
citA Sensor Kinase CitA (transmitter kinase c itA) EP1108790 AX120161
citB Transcription ReguIator CitB (transcriptional regulation protein CitB) EP1108790 AX120163
citE Citrate Lyase EC 4.1.3.6 (citrate lyase) WO0100844 EP1108790 AX065421 AX127146
fda Fructose Bisphosphate Aldolase EC 4.1.2.13 (fructose-1,6-diphosphate zymohexase) People such as von der Osten, Molecular Microbiology 3 (11): 1625-37 (1989) X17313
gluA Glutamate Transport ATP-binding Protein (glutamate transport ATP-is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluB Glutamate-binding Protein (L-glutamic acid is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluC Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluD Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
luxR Transcription Regulator LuxR (transcriptional regulation protein LuxR) WO0100842 EP1108790 AX065953 AX123320
1uxS Histidine Kinase LuxS (histidine kinase LuxS) EP1108790 AX123323 AX127145
lysR1 Transcription Regulator LysR1 (transcriptional regulation protein LysR1) EP1108790 AX064673 AX127144
lysR2 Transcription Activator LysR2 (transcriptional regulation protein LysR2) EP1108790 AX123312
lysR3 Transcription Regulator LysR3 (transcriptional regulation protein LysR3) WO0100842 EP1108790 AX065957 AX127150
menE O-Succinylbenzoic Acid CoA Ligase EC 6.2.1.26 (O-succinyl-phenylformic acid CoA ligase enzyme) WO0100843 EP1108790 AX064599 AX064193 AX127144
mqo Malate-Quinone Oxidoreductase (oxysuccinic acid-benzoquinones-oxydo-reductase) People such as Molenaar, Bur. Journal of Biochemistry 1; 254 (2): 395-403 (1998) AJ224946
pck Phosphoenol Pyruvate Carboxykinase (phosphoenolpyruvate carboxykinase) WO0100844 AJ269506 AX065053
pgi Glucose 6-phosphate Isomerase EC 5.3.1.9 (G-6-P isomerase) EP1087015 EP1108790 AX136015 AX127146
poxB Pyruvate Oxidase EC 1.2.3.3 (pyruvic oxidase) WO0100844 EP1096013 AX064959 AX137665
zwa2 Cell Growth Factor 2 (growth factor-2) EP1106693 EP1108790 AX113822 AX127146
The present invention correspondingly also provides preparation to produce the method for the coryneform bacterium of L-Methionin, comprises
A) separating at least one is produced purpose ORF, gene or the allelic nucleotide sequence of Methionin, comprise alternatively expressing and/or adjustment signal,
B) for the ORF that produces Methionin, gene or allelic 5 ' and 3 ' end the nucleotide sequence of target site is provided, wherein said site is selected from intergenic region, prophage, defective phage or phage assembly,
C) purpose ORF, gene or the allelic nucleotide sequence that preferably will have a target site nucleotide sequence is integrated into and do not duplicate in coryneform bacterium or only carry out in the limited carrier that duplicates,
D) with b) or described nucleotide sequence c) or carrier shift in the coryneform bacterium and
E) separate target site and be integrated with coryneform bacterium according to a) nucleotide sequence.The target site place do not remain in the described microorganism can/make it possible to carry out episomal replication, or can/make it possible to take place swivel base, or give the nucleotide sequence of its antibiotics resistance, for example have the nucleotide sequence of carrier initial point.
The present invention also provides the bacterium according to coryneform bacterium, the especially Corynebacterium of the production L-methionine(Met) of claim 1 and/or L-Threonine.
The present invention also provides the method for preparing L-methionine(Met) and/or L-Threonine according to claim 20.
Open reading-frame (ORF) (ORF), gene or the allelic copy of methionine(Met) " produce " is interpreted as and means, and all have the raising methionine(Met) and produce this effect, preferred endogenous open reading-frame (ORF), gene or allelotrope after enhancings/mistakes expression.
These comprise following open reading-frame (ORF), gene or allelotrope: accBC, accDA, aecD, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, hom, hom except that other FBR, lysC, lysC FBR, metA, metB, metE, metH, metY, msiK, opcA, oxyR, ppc, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T.These summaries and explanation are in table 4.They comprise, the lysC of " feedback " resistance E.C. 2.7.2.4. of especially encoding FBRAllelotrope (referring to table 2) and coding " feedback " resistance homoserine dehydrogenase hom FBRAllelotrope.
" feedback " resistance homoserine dehydrogenase is interpreted as and means, compare with the wild-type form, the inhibition that is caused by Threonine or AHV (pantonine-hydroxypentanoic acid) is had the homoserine dehydrogenase of lower (at least 5% to 10%, 10% to 15% or 10% to 20%) susceptibility.The bacterial strain of producing the L-Threonine contains homoserine dehydrogenase such " feedback " resistance or that desensitize (also referring to people such as Eikmanns the 153rd to 156 page (Antonie vanLeuwenhoek 64:145-163,1993)) usually.
In the 3rd or the 4th site, can integrate open reading-frame (ORF) (ORF), gene or the allelic the 3rd or the 4th copy of producing methionine(Met).Except that other, following open reading-frame (ORF), gene or nucleotide sequence can be used for this: brnE, brnF, brnQ, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, metD, metK, pck, pgi, poxB and zwa2.These summaries and explanation are in table 5.
The site of being mentioned not only comprises the open reading-frame (ORF) mentioned or the coding region of gene certainly, comprise that also being positioned at the upstream is responsible for the zone or the nucleotide sequence of expressing and regulating and control, such as the binding site and the attenuator of ribosome bind site, promotor, modulin binding site, regulation and control Yeast Nucleic Acid.These zones are usually located in coding region upstream 1-800,1-600,1-400,1-200,1-100 or 1-50 the Nucleotide scope.Equally, be positioned at the zone in downstream, in being also included within such as transcription terminator.These zones are usually located in coding region downstream 1-400,1-200,1-100,1-50 or 1-25 the Nucleotide scope.
Use intrachromosomal intergenic region, just do not have the nucleotide sequence of encoding function.At last, be generally comprised within prophage in the karyomit(e) or defective phage or phage assembly and also can be used for this.
Represent the example of the Corynebacterium glutamicum chromosomal region of intergenic region, prophage, defective phage or phage assembly to be shown in table 12 and 13.The position in DNA zone is with reference to Corynebacterium glutamicum ATCC 13032 Genome Atlas shown in EP-A-1108790 or European Molecular Bioglogy Laboratory (EMBL, Heidelberg, Germany and Cambridge, the Britain) database.
With regard to the intergenic region of table 12, it is evident that to those skilled in the art, because different mutually and handle between wild-type and the wild type strain, can use according to the present invention and contain that to have at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and do not have encoding function with the described nucleotide sequence of table 12 be gene or allelic zone according to the sudden change of being accepted.
With regard to the DNA zone of the coding prophage, defective phage or the phage assembly that are selected from table 13, it is evident that to those skilled in the art, since different mutually and handle between wild-type and the wild type strain according to the sudden change of being accepted, can use according to the present invention and contain the zone that has at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and coding prophage, defective phage or phage assembly with the described nucleotide sequence of table 13.
Table 4
Produce open reading-frame (ORF), gene and the allelotrope of methionine(Met)
Title Coded enzyme or proteinic explanation Reference Accession number
AccBC Acyl group-CoA carboxylase EC 6.3.4.14 (acyl group-CoA carboxylase) People such as J_ger, Archives of Microbiology (1996) 166:76-82 EP1108790; WO0100805 U35023 AX123524 AX066441
AccDA Acetyl-CoA carboxylase EC 6.4.1.2 EP1055725 EP1108790 AX121013
(acetyl-CoA carboxylase) WO0100805 AX066443
AecD Cystathionine beta-Lyase EC 4.4.1.8 (cystathionine beta-lyase) People such as Rossol, Journal of Bacteriology 174:2968-2977 (1992) M89931
CstA Carbon Starvation Protein A (the hungry albumin A of carbon) EP1108790 WO0100804 AX120811 AX066109
CysD Sulfate Adenylyltransferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) EP1108790 AX123177
CysE Serine Acetyltransferase EC 2.3.1.30 (serine acetyltransferase) EP1108790 WO0100843 AX122902 AX063961
CysH 3 '-Phosphoadenyl Sulfate Reductase EC 1.8.99.4 (3'-phosphoadenosine-5'-phosphosulfate reductase enzyme) EP1108790 WO0100842 AX123178 AX066001
CysK Cysteine Synthase EC 4.2.99.8 (cysteine synthase) EP1108790 WO0100843 AX122901 AX063963
CysN Sulfate Adenylyltransferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) EP1108790 AX123176 AX127152
CysQ Transport protein CysQ (translocator cysQ) EP1108790 WO0100805 AX127145 AX066423
Dps DNA Protection Protein (albumen of between hunger period, protecting) EP1108790 AX127153
Eno Enolase EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) People such as EP1108790 WO0100844 EP1090998 Hermann, Electrophoresis 19:3217-3221 (1998) AX127146 AX064945 AX136862
Fda Fructose Bisphosphate Aldolase EC 4.1.2.13 (fructose-bis phosphate aldolase) People such as van der Osten, Molecular Microbiology 3:1625-1637 (1989) X17313
Gap Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) People such as EP1108790 WO0100844 Eikmanns, Journal of Bacteriology 174:6076-6086 (1992) AX127148 AX064941 X59403
gap2 Glyceraldehyde 3-Phosphate Dehydrogenase EP1108790 WO0100844 AX127146 AX064939
EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2)
Gdh Glutamate Dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) People such as EP1108790 WO0100844 Boermann, Molecular Microbiology 6:317-326 (1992); People such as Guyonvarch, NCBI AX127150 AX063811 X59404 X72855
GlyA Glycine/Serine Hydroxymethyltransferase EC 2.1.2.1 (glycine/serine hydroxymethylase) EP1108790 AX127146 AX121194
Gnd 6-Phosphogluconate Dehydrogenase EC 1.1.1.44 (6-Phosphogluconic dehydrogenase) EP1108790 WO0100844 AX127147 AX121689 AX065125
Hom Homoserine Dehydrogenase EC 1.1.1.3 (homoserine dehydrogenase) People such as Peoples, Molecular Microbiology 2:63-72 (1988) Y00546
hom FBR Homoserine Dehydrogenase feedback resistant (fbr) EC 1.1.1.3 (homoserine dehydrogenase fbr) People such as Reinscheid, Journal of Bacteriology 173:3228-30 (1991)
LysC Aspartate Kinase EC 2.7.2.4 (E.C. 2.7.2.4.) People such as BP1108790 WO0100844 Kalinowski, Molecular Microbiology 5:1197-204 (1991) AX120365 AX063743 X57226
lysC FBR Aspartate Kinase feedback resistant (fbr) EC 2.7.2.4 (E.C. 2.7.2.4. fbr) Referring to table 2
MetA Homoserine Acetyltransferase EC 2.3.1.31 (homoserine acetyltransferase) People such as Park, Molecular Cells 8:286-94 (1998) AF052652
MetB Cystathionine γ-Lyase EC 4.4.1.1 (cystathionine Gamma-lyase) People such as Hwang, Molecular Cells 9:300-308 (1999) AF126953
MetE Homocysteine Methyltransferase EC 2.1.1.14 (homocysteine methyl transferase) EP1108790 AX127146 AX121345
MetH Homocysteine Methyltransferase (Vitamin B12-dependent) EP1108790 AX127148 AX121747
EC 2.1.1.14 (homocysteine methyl transferase)
MetY Acetylhomoserine Sulfhydrolase (ethanoyl homoserine sulphur lytic enzyme) EP1108790 AX120810 AX127145
MsiK Sugar Importer (various saccharides input albumen) EP1108790 AX120892
OpcA Glucose 6-phosphate Dehydrogenase (subunit of glucose-6-phosphate dehydrogenase (G6PD)) WO0104325 AX076272
OxyR Transcription Regulator (transcriptional regulation protein) EP1108790 AX122198 AX127149
ppc FBR Phosphoenol Pyruvate Carboxylase feedback resistent EC 4.1.1.31 (Phosphoenolpyruvate carboxylase of feedback resistance) EP0723011 WO0100852
Ppc Phosphoenol Pyruvate Carboxylase EC 4.1.1.31 (Phosphoenolpyruvate carboxylase) People such as EP1108790 O ' Reagan, Gene 77 (2): 237-251 (1989) AX127148 AX123554 M25819
Pgk Phosphoglycerate Kinase EC 2.7.2.3 (phosphoglyceric kinase) EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174：6076-6086(1992) AX121838 AX127148 AX064943 X59403
PknA Protein Kinase A (protein kinase A) EP1108790 AX120131 AX120085
PknB Protein Kinase B (protein kinase B) EP1108790 AX120130 AX120085
PknD Protein Kinase D (protein kinase D) EP1108790 AX127150 AX122469 AX122468
PknG Protein Kinase G (protein kinase G) EP1108790 AX127152 AX123109
PpsA Phosphoenol Pyruvate Synthase EC 2.7.9.2 (phosphoenolpyruvate synthase) EP1108790 AX127144 AX120700 AX122469
PtsH Phosphotransferase System Protein H EC 2.7.1.69 (phosphotransferase system component H) EP1108790 WO0100844 AX122210 AX127149 AX069154
PtsI Phosphotransferase System Enzyme I EC 2.7.3.9 (phosphotransferase system enzyme I) EP1108790 AX122206 AX127149
PtsM Glucose-specific Phosphotransferase System Enzyme II EC 2.7.1.69 People such as Lee, FEMS Microbiology Letters 119 (1-2): 137-145 L18874
(glucose phosphotransferase system enzyme II) (1994)
Pyc Pyruvate Carboxylase EC 6.4.1.1 (pyruvate carboxylase) People such as WO9918228 Peters-Wendisch, Microbiology 144:915-927 (1998) A97276 Y09548
Pyc P458s Pyruvate Carboxylase EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S EP1108790
SigC Sigma Factor C EC 2.7.7.6 (the outer function substituted type Sigma Factors C of kytoplasm) EP1108790 AX120368 AX120085
SigD RNA Polymerase Sigma Factor D EC 2.7.7.6 (RNA polymerase Sigma Factors) EP1108790 AX120753 AX127144
SigE Sigma Factor E EC 2.7.7.6 (the outer function substituted type Sigma Factors E of kytoplasm) EP1108790 AX127146 AX121325
SigH Sigma Factor H EC 2.7.7.6 (Sigma Factors SigH) EP1108790 AX127145 AX120939
SigM Sigma Factor M EC 2.7.7.6 (Sigma Factors SigM) EP1108790 AX123500 AX127153
Tal Transaldolase EC 2.2.1.2 (transaldolase) WO0104325 AX076272
ThyA Thymidylate Synthase EC 2.1.1.45 (thymidylate synthase) EP1108790 AX121026 AX127145
Tkt Transketolase EC 2.2.1.1 (transketolase) People such as Ikeda, NCBI AB023377
Tpi Triose Phosphate Isomerase EC 5.3.1.1 (triose-phosphate isomerase) Eikmanns，Journal of Bacteriology 174：6076-6086(1992) X59403
zwa1 Cell Growth Factor 1 (somatomedin 1) EP1111062 AX133781
Zwf Glucose 6-phosphate 1-Dehydrogenase EC 1.1.1.49 (G-6-P 1-desaturase) EP1108790 WO0104325 AX127148 AX121827 AX076272
Zwf A213T Glucose 6-phosphate 1-Dehydrogenase EC 1.1.1.49 (G-6-P 1-desaturase) amino acid exchange A213T EP1108790
Table 5
Be used to integrate open reading-frame (ORF), gene and the allelic target site of producing methionine(Met)
The gene title Coded enzyme and proteinic explanation Reference Accession number
BrnE Transporter of branched-chain amino acids (branched-chain amino acid translocator) EP1096010 AX137709 AX137714
BrnF Transporter of branched-chain amino acids (branched-chain amino acid translocator) EP1096010 AX137709 AX137714
BrnQ Carrier protein of branched-chain amino acids (branched-chain amino acid transports system's carrier proteins) People such as Tauch, Archives of Microbiology 169 (4): 303-12 (1998) WO0100805 EP1108790 M89931 AX066841 AX127150
ccpA1 Catabolite Control Protein (catabolite control albumin A 1) WO0100844 EP1108790 AX065267 AX127147
ccpA2 Catabolite Control Protein (catabolite control albumin A 2) WO0100844 EP1108790 AX065267 AX121594
citA Sensor Kinase CitA (transmitter kinase c itA) EP1108790 AX120161
citB Transcription Regulator CitB (transcriptional regulation protein CitB) EP1108790 AX120163
citE Citrate Lyase EC 4.1.3.6 (citrate lyase) WO0100844 EP1108790 AX065421 AX127146
ddh Diaminopimelate Dehydrogenase EC 1.4.1.16 (diaminopimelate dehydrogenase) People such as Ishino, Nucleic Acids Research 15:3917 (1987) EP1108790 S07384 AX127152
gluA Glutamate Transport ATP-binding Protein (glutamate transport ATP-is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluB Glutamate-binding Protein (L-glutamic acid is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluC Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluD Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
luxR Transcription Regulator LuxR (transcriptional regulation protein LuxR) WO0100842 EP1108790 AX065953 AX123320
luxS Histidine Kinase LuxS (histidine kinase LuxS) EP1108790 AX123323 AX127145
lysR1 Transcription Regulator LysR1 (transcriptional regulation protein LysR1) EP1108790 AX064673 AX127144
lysR2 Transcription Activator LysR2 (transcriptional regulation protein LysR2) EP1108790 AX123312
lysR3 Transcription Regulator LysR3 (transcriptional regulation protein LysR3) WO0100842 EP1108790 AX065957 AX127150
menE O-Succinylbenzoic Acid CoA Ligase EC 6.2.1.26 (O-succinyl-phenylformic acid CoA ligase enzyme) WO0100843 EP1108790 AX064599 AX064193 AX127144
metD Transcription Regulator MetD (transcriptional regulation protein MetD) EP1108790 AX123327 AX127153
metK Methionine Adenosyl Transferase EC 2.5.1.6 (S-adenosylmethionine synthetic enzyme) WO0100843 EP1108790 AX063959 AX127148
pck Phosphoenol Pyruvate Carboxykinase (phosphoenolpyruvate carboxykinase) WO0100844 AJ269506 AX065053
pgi Glucose 6-Phosphate Isomerase EC 5.3.1.9 (G-6-P isomerase) EP1087015 EP1108790 AX136015 AX127146
poxB Pyruvate Oxidase EC 1.2.3.3 (pyruvic oxidase) WO0100844 EP1096013 AX064959 AX137665
zwa2 Ce11 Growth Factor 2 (growth factor-2) EP1106693 EP1108790 AX113822 AX127146
Open reading-frame (ORF) (ORF), gene or the allelic copy of Threonine " produce " is interpreted as and means, and all have raising Methionin and produce this effect, preferred endogenous open reading-frame (ORF), gene or allelotrope after enhancings/mistakes expression.
These comprise following open reading-frame (ORF), gene or allelotrope: accBC, accDA, cstA, cysD, cysE, cysH, cysI, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, hom, hom except that other FBR, lysC, lysC FBR, msiK, opcA, oxyR, ppc, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, thrB, thrC, thrE, zwa1, zwf and zwfA213T.These summaries and explanation are in table 6.They comprise, the lysC of " feedback " resistance E.C. 2.7.2.4. of especially encoding FBRAllelotrope (referring to table 2) and coding " feedback " resistance homoserine dehydrogenase hom FBRAllelotrope.
" feedback " resistance homoserine dehydrogenase is interpreted as and means, compare with the wild-type form, the inhibition that is caused by Threonine or AHV (pantonine-hydroxypentanoic acid) is had the homoserine dehydrogenase of lower (at least 5% to 10%, 10% to 15% or 10% to 20%) susceptibility.The bacterial strain of producing the L-Threonine contains homoserine dehydrogenase such " feedback " resistance or that desensitize (also referring to people such as Eikmanns the 153rd to 156 page (Antonie vanLeuwenhoek 64:145-163,1993)) usually.
In the 3rd or the 4th site, can integrate open reading-frame (ORF) (ORF), gene or the allelic the 3rd or the 4th copy of producing Threonine.Except that other, following open reading-frame (ORF), gene or nucleotide sequence can be used as integration site: ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, ilvBN, ilvC, ilvD, luxR, luxS, lysR1, lysR2, lysR3, mdh, menE, metA, metD, pck, poxB, sigB and zwa2.These summaries and explanation are in table 7.
The site of being mentioned not only comprises the open reading-frame (ORF) mentioned or the coding region of gene certainly, comprise that also being positioned at the upstream is responsible for the zone or the nucleotide sequence of expressing and regulating and control, such as the binding site and the attenuator of ribosome bind site, promotor, modulin binding site, regulation and control Yeast Nucleic Acid.These zones are usually located in coding region upstream 1-800,1-600,1-400,1-200,1-100 or 1-50 the Nucleotide scope.Equally, be positioned at the zone in downstream, in being also included within such as transcription terminator.These zones are usually located in coding region downstream 1-400,1-200,1-100,1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region, just not have the nucleotide sequence of encoding function.At last, be generally comprised within prophage in the karyomit(e) or defective phage or phage assembly and also can be used for this.
Represent the example of the Corynebacterium glutamicum chromosomal region of intergenic region, prophage, defective phage or phage assembly to be shown in table 12 and 13.The position in DNA zone is with reference to Corynebacterium glutamicum ATCC 13032 Genome Atlas shown in EP-A-1108790 or European Molecular Bioglogy Laboratory (EMBL, Heidelberg, Germany and Cambridge, the Britain) database.
With regard to the intergenic region of table 12, it is evident that to those skilled in the art, because different mutually and handle between wild-type and the wild type strain, can use according to the present invention and contain that to have at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and do not have encoding function with the described nucleotide sequence of table 12 be gene or allelic zone according to the sudden change of being accepted.
With regard to the DNA zone of the coding prophage, defective phage or the phage assembly that are selected from table 13, it is evident that to those skilled in the art, since different mutually and handle between wild-type and the wild type strain according to the sudden change of being accepted, can use according to the present invention and contain the zone that has at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and coding prophage, defective phage or phage assembly with the described nucleotide sequence of table 13.
Table 6
Produce open reading-frame (ORF), gene and the allelotrope of Threonine
Title Coded enzyme or proteinic explanation Reference Accession number
accBC Acyl group-CoA carboxylase EC 6.3.4.14 (acyl group-CoA carboxylase) People such as J_ger, Archives of Microbiology 166:76-82 (1996) EP1108790 WO0100805 U35023 AX123524 AX066441
accDA Acetyl-CoA carboxylase EC 6.4.1.2 (acetyl-CoA carboxylase) EP1055725 EP1108790 WO0100805 AX121013 AX066443
cstA Carbon Starvation Protein A (the hungry albumin A of carbon) EP1108790 WO0100804 AX120811 AX066109
cysD Sulfate Adenylyltransferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) EP1108790 AX123177
cysE Serine Acetyltransferase EC 2.3.1.30 (serine acetyltransferase) EP1108790 WO0100843 AX122902 AX063961
cysH 3 '-Phosphoadenyl Sulfate Reductase EC 1.8.99.4 (3'-phosphoadenosine-5'-phosphosulfate reductase enzyme) EP1108790 WO0100842 AX123178 AX066001
cysK Cysteine Synthase EC 4.2.99.8 (cysteine synthase) EP1108790 WO0100843 AX122901 AX063963
cysN Sulfate Adenylyltransferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) EP1108790 AX123176 AX127152
cysQ Transport protein CysQ (translocator cysQ) EP1108790 WO0100805 AX127145 AX066423
dps DNA Protection Protein (albumen of between hunger period, protecting) EP1108790 AX127153
eno Enolase EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) People such as EP1108790 WO0100844 EP1090998 Hermann, Electrophoresis 19:3217-3221 (1998) AX127146 AX064945 AX136862
fda Fructose Bisphosphate Aldolase EC 4.1.2.13 (fructose-bis phosphate aldolase) People such as van der Osten, Molecular Microbiology 3:1625-1637 (1989) X17313
gap Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) People such as EP1108790 WO0100844 Eikmanns, Journal of Bacteriology 174:6076-6086 (1992) AX127148 AX064941 X59403
gap2 Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) EP1108790 WO0100844 AX127146 AX064939
gdh Glutamate Dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) People such as EP1108790 WO0100844 Boermann, Molecular Microbiology 6:317-326 (1992); People such as Guyonvarch, NCBI AX127150 AX063811 X59404 X72855
gnd 6-Phosphogluconate Dehydrogenase EC 1.1.1.44 (6-Phosphogluconic dehydrogenase) EP1108790 WO0100844 AX127147 AX121689 AX065125
hom Homoserine Dehydrogenase EC 1.1.1.3 (homoserine dehydrogenase) People such as Peoples, Molecular Microbiology 2:63-72 Y00546
(1988)
hom FBR Homoserine Dehydrogenase feedback resistant (fbr) EC 1.1.1.3 (homoserine dehydrogenase fbr) People such as Reinscheid, Journal of Bacteriology 173:3228-30 (1991)
lysC Aspartate Kinase EC 2.7.2.4 (E.C. 2.7.2.4.) People such as EP1108790 WO0100844 Kalinowski, Molecular Microbiology 5:1197-204 (1991) AX120365 AX063743 X57226
lysC FBR Aspartate Kinase feedback resistent (fbr) EC 2.7.2.4 (E.C. 2.7.2.4. fbr) Referring to table 2
msiK Sugar Importer (various saccharides input albumen) EP1108790 AX120892
opcA Glucose 6-Phosphate Dehydrogenase (subunit of glucose-6-phosphate dehydrogenase (G6PD)) WO0104325 AX076272
oxyR Transcription Regulator (transcriptional regulation protein) EP1108790 AX122198 AX127149
ppc FBR Phosphoenol Pyruvate Carboxylase feedback resistent EC 4.1.1.31 (Phosphoenolpyruvate carboxylase of feedback resistance) EP0723011 WO0100852
ppc Phosphoenol Pyruvate Carboxylase EC 4.1.1.31 (Phosphoenolpyruvate carboxylase) People such as EP1108790 O ' Reagan, Gene 77 (2): 237-251 (1989) AX127148 AX123554 M25819
pgk Phosphoglycerate Kinase EC 2.7.2.3 (phosphoglyceric kinase) EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174：6076-6086(1992) AX121838 AX127148 AX064943 X59403
pknA Protein Kinase A (protein kinase A) EP1108790 AX120131 AX120085
pknB Protein Kinase B (protein kinase B) EP1108790 AX120130 AX120085
pknD Protein Kinase D (protein kinase D) EP1108790 AX127150 AX122469 AX122468
pknG Protein Kinase G (protein kinase G) EP1108790 AX127152 AX123109
ppsA Phosphoenol Pyruvate Synthase EC 2.7.9.2 (phosphoenolpyruvate synthase) EP1108790 AX127144 AX120700 AX122469
ptsH Phosphotransferase System Protein H EC 2.7.1.69 (phosphotransferase system component H) EP1108790 WO0100844 AX122210 AX127149 AX069154
ptsI Phosphotransferase System Enzyme I EC 2.7.3.9 (phosphotransferase system enzyme I) EP1108790 AX122206 AX127149
ptsM Glukose-Specific Phosphotransferase System Enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) People such as Lee, FEMS Microbiology Letters 119 (1-2): 137-145 (1994) L18874
pyc Pyruvate Carboxylase EC 6.4.1.1 (pyruvate carboxylase) People such as WO9918228 Peters-Wendisch, Microbiology 144:915-927 (1998) A97276 Y09548
pyc P458S Pyruvate Carboxylase EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S EP1108790
sigC Sigma Factor C EC 2.7.7.6 (the outer function substituted type factor C of kytoplasm) EP1108790 AX120368 AX120085
sigD RNA Polymerase Sigma Factor D EC 2.7.7.6 (RNA polymerase Sigma Factors) EP1108790 AX120753 AX127144
sigE Sigma Factor E EC 2.7.7.6 (the outer function substituted type Sigma Factors E of kytoplasm) EP1108790 AX127146 AX121325
sigH Sigma Factor H EC 2.7.7.6 (Sigma Factors SigH) EP1108790 AX127145 AX120939
sigM Sigma Factor M EC 2.7.7.6 (Sigma Factors SigM) EP1108790 AX123500 AX127153
tal Transaldolase EC 2.2.1.2 (transaldolase) WO0104325 AX076272
thrB Homoserine Kinase EC 2.7.1.39 (homoserine kinase) People such as Peoples, Molecular Microbiology 2:63-72 (1988) Y00546
thrC Threonine Synthase EC 4.2.99.2 People such as Han, Molecular Microbiology X56037
(threonine synthase) 4：1693-1702(1990)
thrE Threonine Exporter (Threonine output carrier) EP1085091 AX137526
thyA Thymidylate Synthase EC 2.1.1.45 (thymidylate synthase) EP1108790 AX121026 AX127145
tkt Transketolase EC 2.2.1.1 (transketolase) People such as Ikeda, NCBI AB023377
tpi Triose phosphate Isomerase EC 5.3.1.1 (triose-phosphate isomerase) Eikmanns，Journal of Bacteriology 174：6076-6086(1992) X59403
zwa1 Cell Growth Factor 1 (somatomedin 1) EP1111062 AX133781
zwf Glucose 6-Phosphate 1-Dehydrogenase EC 1.1.1.49 (G-6-P 1-desaturase) EP1108790 WO0104325 AX127148 AX121827 AX076272
zwf A213T Glucose 6-Phosphate 1-Dehydrogenase EC 1.1.1.4 9 (G-6-P 1-desaturase) amino acid exchange A213T EP1108790
Table 7
Be used to integrate open reading-frame (ORF), gene and the allelic target site of producing Threonine
The gene title Coded enzyme or proteinic explanation Reference Accession number
ccpA1 Catabolite Control Protein (catabolite control albumin A 1) WO0100844 EP1108790 AX065267 AX127147
ccpA2 Catabo1ite Control Protein (catabolite control albumin A 2) WO0100844 EP1108790 AX065267 AX121594
citA Sensor Kinase CitA (transmitter kinase c itA) EP1108790 AX120161
citB Transcription Regulator CitB (transcriptional regulation protein CitB) EP1108790 AX120163
citE Citrate Lyase EC 4.1.3.6 (citrate lyase) WO0100844 EP1108790 AX065421 AX127146
ddh Diaminopimelate Dehydrogenase EC 1.4.1.16 (diaminopimelate dehydrogenase) People such as Ishino, Nucleic Acids Research 15:3917 (1987) EP1108790 S07384 AX127152
gluA Glutamate Transport ATP-binding Protein (glutamate transport ATP-is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluB Glutamate-binding Protein (L-glutamic acid is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluC Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluD Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
glyA Glycine Hydroxymethyltransferase EC 2.1.2.1 (glycine hydroxymethyltransferase) WO0100843 AX063861 AF327063
ilvA Threonine Dehydratase EC 4.2.1.16 (threonine dehydra(ta)se) People such as M_ckel, Journal of Bacteriology 174 (24), 8065-8072 (1992) EP1108790 A47044 L01508 AX127150
ilvBN Acetolactate Synthase EC 4.1.3.18 (acetolactate synthase) People such as Keilhauer, Journal of Bacteriology 175 (17): 5595-603 (1993) EP1108790 L09232 AX127147
ilvC Reductoisomerase EC 1.1.1.86 (ketol-acid reductoisomerase) People such as Keilhauer, Journal of Bacteriology 175 (17): 5595-603 (1993) EP1108790 C48648 AX127147
ilvD Dihydroxy-acid Dehydratase EC 4.2.1.9 (dihydroxyacid dehydratase) EP1006189 AX136925
luxR Transcription Regulator LuxR (transcriptional regulation protein LuxR) WO0100842 EP1108790 AX065953 AX123320
luxS Histidine Kinase LuxS (histidine kinase LuxS) EP1108790 AX123323 AX127153
lysR1 Transcription Regulator LysR1 (transcriptional regulation protein LysR1) EP1108790 AX064673 AX127144
lysR2 Transcription Activator LysR2 EP1108790 AX123312
(transcriptional regulation protein LysR2)
lysR3 Transcription Regulator LysR3 (transcriptional regulation protein LysR3) WO0100842 EP1108790 AX065957 AX127150
mdh Malate Dehydrogenase EC 1.1.1.37 (malate dehydrogenase (malic acid dehydrogenase)) WO0100844 AX064895
menE O-Succinylbenzoic Acid CoA Ligase EC 6.2.1.26 (O-succinyl-phenylformic acid CoA ligase enzyme) WO0100843 EP1108790 AX064599 AX064193 AX127144
metA Homoserine O-Acetyltransferase EC 2.3.1.31 (homoserine O-acetyltransferase) People such as Park, Molecular Cells 30; 8 (3): 286-94 (1998) WO0100843 EP1108790 AX063895 AX127145
metD Transcription Regulator MetD (transcriptional regulation protein MetD) EP1108790 AX123327 AX127153
pck Phosphoenol Pyruvate Carboxykinase (phosphoenolpyruvate carboxykinase) WO0100844 AJ269506 AX065053
poxB Pyruvate Oxidase EC 1.2.3.3 (pyruvic oxidase) WO0100844 EP1096013 AX064959 AX137665
sigB RNA Polymerase Transcription Factor (the rna polymerase transcribe factor) EP1108790 AX127149
zwa2 Cell Growth Factor 2 (growth factor-2) EP1106693 EP1108790 AX113822 AX127146
The present invention correspondingly also provides preparation to produce the method for the coryneform bacterium of L-methionine(Met) and/or L-Threonine, comprises
A) separating at least one is produced purpose ORF, gene or the allelic nucleotide sequence of methionine(Met) or Threonine, comprise alternatively expressing and/or adjustment signal,
B) for ORF, gene or allelic 5 ' and 3 ' end the nucleotide sequence of target site is provided, wherein said site is selected from intergenic region, prophage, defective phage or phage assembly,
C) preferably described purpose ORF, gene or the allelic nucleotide sequence that has the target site nucleotide sequence is integrated into and in coryneform bacterium, do not duplicate or only carry out in the limited carrier that duplicates,
D) with b) or described nucleotide sequence c) or carrier shift in the coryneform bacterium and
F) separate target site and be integrated with coryneform bacterium according to a) nucleotide sequence.
Preferred target site place do not remain in the described microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/nucleotide sequence that makes it possible to that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.
The present invention also provides the bacterium according to coryneform bacterium, the especially Corynebacterium of the production L-Xie Ansuan of claim 1.
The present invention also provides the method for preparing the L-Xie Ansuan according to claim 20.
" open reading-frame (ORF) (ORF), gene or the allelic copy of production Xie Ansuan " is interpreted as and means, and all have the Xie Ansuan of raising and produce open reading-frame (ORF), gene or the allelotrope of this effect after enhancing/mistake is expressed.
These comprise following open reading-frame (ORF), gene or allelotrope: brnE, brnF, brnEF, cstA, cysD, dps, eno, fda, gap, gap2, gdh, ilvB, ilvN, ilvBN, ilvC, ilvD, ilvE, msiK, pgk, ptsH, ptsI, ptsM, sigC, sigD, sigE, sigH, sigM, tpi, zwa1 except that other.These summaries and explanation are in table 8.They especially comprise the ilvBN allelotrope of coding Xie Ansuan-resistance acetolactate synthase.
" feedback " resistance acetolactate synthase (=acetohydroxy acid synthetase) is interpreted as and means, compare with the wild-type form, the inhibition that is caused by Xie Ansuan or Xie Ansuan analogue 2-thiazole L-Ala (EPappln.No.03014640.1) is at least had the acetolactate synthase of lower (at least 10% to 15% or 10% to 20%) susceptibility.
In the 3rd or the 4th site, can integrate open reading-frame (ORF) (ORF), gene or the allelic the 3rd or the 4th copy of producing Xie Ansuan.Except that other, following open reading-frame (ORF), gene or nucleotide sequence can be used for this: aecD, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lySR1, lySR2, lysR3, panB, panC, poxB and zwa2.These summaries and explanation are in table 9.
The site of being mentioned not only comprises the open reading-frame (ORF) mentioned or the coding region of gene certainly, comprise that also being positioned at the upstream is responsible for the zone or the nucleotide sequence of expressing and regulating and control, such as the binding site and the attenuator of ribosome bind site, promotor, modulin binding site, regulation and control Yeast Nucleic Acid.These zones are usually located in coding region upstream 1-800,1-600,1-400,1-200,1-100 or 1-50 the Nucleotide scope.Equally, be positioned at the zone in downstream, in being also included within such as transcription terminator.These zones are usually located in coding region downstream 1-400,1-200,1-100,1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region, just not have the nucleotide sequence of encoding function.At last, be generally comprised within prophage in the karyomit(e) or defective phage or phage assembly and can be used for this.
Represent the example of the Corynebacterium glutamicum chromosomal region of intergenic region, prophage, defective phage or phage assembly to be shown in table 12 and 13.The position in DNA zone is with reference to Corynebacterium glutamicum ATCC 13032 Genome Atlas shown in EP-A-1108790 or European Molecular Bioglogy Laboratory (EMBL, Heidelberg, Germany and Cambridge, the Britain) database.
With regard to the intergenic region of table 12, it is evident that to those skilled in the art, because different mutually and handle between wild-type and the wild type strain, can use according to the present invention and contain that to have at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and do not have encoding function with the described nucleotide sequence of table 12 be gene or allelic zone according to the sudden change of being accepted.
With regard to the DNA zone of the coding prophage, defective phage or the phage assembly that are selected from table 13, it is evident that to those skilled in the art, since different mutually and handle between wild-type and the wild type strain according to the sudden change of being accepted, can use according to the present invention and contain the zone that has at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% conforming nucleotide sequence and coding prophage, defective phage or phage assembly with the described nucleotide sequence of table 13.
Table 8
Produce open reading-frame (ORF), gene and the allelotrope of Xie Ansuan
Title Coded enzyme or proteinic explanation Reference Accession number
brnEF Export of branched-chain amino acids (branched-chain amino acid output) People such as EP1096010 Kennerknecht, AF454053
NCBI
cstA Carbon Starvation Protein A (the hungry albumin A of carbon) EP1108790 WO0100804 AX120811 AX066109
dps DNA Protection Protein (albumen of between hunger period, protecting) EP1108790 AX127153
eno Enolase EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) People such as EP1108790 WO0100844 EP1090998 Hermann, Electrophoresis 19:3217-3221 (1998) AX127146 AX064945 AX136862
fda Fructose Bisphosphate Aldolase EC 4.1.2.13 (fructose-bis phosphate aldolase) People such as van der Osten, Molecular Microbiology 3:1625-1637 (1989) X17313
gap Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) People such as EP1108790 WO0100844 Eikmanns, Journal of Bacteriology 174:6076-6086 (1992) AX127148 AX064941 X59403
gap2 Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) EP1108790 WO0100844 AX127146 AX064939
gdh Glutamate Dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) People such as EP1108790 WO0100844 Boermann, Molecular Microbiology 6:317-326 (1992); People such as Guyonvarch, NCBI AX127150 AX063811 X59404 X72855
ilvBN Acetolactate Synthase EC 4.1.3.18 (acetolactate synthase) People such as Keilhauer, Journal of Bacteriology 175 (17): 5595-603 (1993) EP1108790 L09232 AX127147
ilvC Isomeroreductase EC 1.1.1.86 (acetohydroxy acid isomeroreductase) People such as Keilhauer, Journal of Bacteriology 175 (17): 5595-603 C48648 AX127147
(1993) BP1108790
ilvD Dihydroxy-acid Dehydratase EC 4.2.1.9 (dihydroxyacid dehydratase) EP1006189 AX136925
ilvE Transaminase B EC 2.6.1.42 (Transaminase B) EP1108790 AX127150 AX122498
msiK Sugar Importer (various saccharides input albumen) EP1108790 AX120892
pgk Phosphoglycerate Kinase EC 2.7.2.3 (phosphoglyceric kinase) EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174：6076-6086 (1992) AX121838 AX127148 AX064943 X59403
ptsH Phosphotransferase System Protein H EC 2.7.1.69 (phosphotransferase system component H) EP1108790 WO0100844 AX122210 AX127149 AX069154
ptsI Phosphotransferase System Enzyme I EC 2.7.3.9 (phosphotransferase system enzyme I) EP1108790 AX122206 AX127149
ptsM Glucose-specific Phosphotransferase System Enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) People such as Lee, FEMS Microbiology Letters 119 (1-2): 137-145 (1994) L18874
sigC Sigma Factor C EC 2.7.7.6 (the outer function substituted type Sigma Factors C of kytoplasm) EP1108790 AX120368 AX120085
sigD RNA Polymerase Sigma Factor D EC 2.7.7.6 (RNA polymerase Sigma Factors) EP1108790 AX120753 AX127144
sigE Sigma Factor E EC 2.7.7.6 (the outer function substituted type Sigma Factors E of kytoplasm) EP1108790 AX127146 AX121325
sigH Sigma Factor H EC 2.7.7.6 (Sigma Factors SigH) EP1108790 AX127145 AX120939
sigM Sigma Factor M EC 2.7.7.6 (Sigma Factors SigM) EP1108790 AX123500 AX127153
tpi Triose Phosphate Isomerase EC 5.3.1.1 Eikmanns，Journal of Bacteriology X59403
(triose-phosphate isomerase) 174：6076-6086 (1992)
zwal Cell Growth Factor 1 (somatomedin 1) EP1111062 AX133781
Table 9
Be used to integrate open reading-frame (ORF), gene and the allelic target site of producing Xie Ansuan
The gene title Coded enzyme or proteinic explanation Reference Accession number
aecD Beta C-S Lyase EC 2.6.1.1 (β C-S lyase) People such as Rossol, Journal of Bacteriology 174 (9): 2968-77 (1992) M89931
ccpA1 Catabolite Control Protein (catabolite control albumin A 1) WO0100844 EP1108790 AX065267 AX127147
ccpA2 Catabolite Control Protein (catabolite control albumin A 2) WO0100844 EP1108790 AX065267 AX121594
citA Sensor Kinase CitA (transmitter kinase c itA) EP1108790 AX120161
citB Transcription Regulator CitB (transcriptional regulation protein CitB) EP1108790 AX120163
citE Citrate Lyase EC 4.1.3.6 (citrate lyase) WO0100844 EP1108790 AX065421 AX127146
ddh Diaminopimelate Dehydrogenase EC 1.4.1.16 (diaminopimelate dehydrogenase) People such as Ishino, Nucleic Acids Research 15:3917 (1987) EP1108790 S07384 AX127152
gluA Glutamate Transport ATP-binding Protein (glutamate transport ATP-is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluB Glutamate-binding Protein (L-glutamic acid is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluC Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluD Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
glyA Glycine Hydroxymethyltransferase EC 2.1.2.1 (glycine hydroxymethyltransferase) WO0100843 AX063861 AF327063
ilvA Threonine Dehydratase EC 4.2.1.16 (threonine dehydra(ta)se) People such as M_ckel, Journal of Bacteriology 174 (24), 8065-8072 (1992) EP1108790 A47044 L01508 AX127150
luxR Transcription Regulator LuxR (transcriptional regulation protein LuxR) WO0100842 EP1108790 AX065953 AX123320
lysR1 Transcription Regulator LysR1 (transcriptional regulation protein LysR1) EP1108790 AX064673 AX127144
lysR2 Transcription Activator LysR2 (transcriptional regulation protein LysR2) EP1108790 AX123312
lysR3 Transcription Regulator LysR3 (transcriptional regulation protein LysR3) WO0100842 EP1108790 AX065957 AX127150
panB Ketopantoate Hydroxymethyltransferase EC 2.1.2.11 (ketopantoic acid hydroxymethyl transferases) US6177264 X96580
panC Pantothenate Synthetase EC 6.3.2.1 (pantothenate synthetase) US6177264 X96580
poxB Pyruvate Oxidase EC 1.2.3.3 (pyruvic oxidase) WO0100844 EP1096013 AX064959 AX137665
zwa2 Cell Growth Factor 2 (growth factor-2) EP1106693 EP1108790 AX113822 AX127146
The present invention correspondingly also provides preparation to produce the method for the coryneform bacterium of L-Xie Ansuan, comprises
A) separating at least one is produced purpose ORF, gene or the allelic nucleotide sequence of Xie Ansuan, comprise alternatively expressing and/or adjustment signal,
B) for ORF, gene or allelic 5 ' and 3 ' end the nucleotide sequence of target site is provided, wherein said site is selected from intergenic region, prophage, defective phage or phage assembly,
C) purpose ORF, gene or the allelic nucleotide sequence that preferably will have a target site nucleotide sequence is integrated into and do not duplicate in coryneform bacterium or only carry out in the limited carrier that duplicates,
D) with b) or described nucleotide sequence c) or carrier shift in the coryneform bacterium and
E) separate target site and be integrated with coryneform bacterium according to a) nucleotide sequence.The target site place do not remain in the microorganism can/make it possible to carry out episomal replication, or can/make it possible to take place swivel base, or give the nucleotide sequence of its antibiotics resistance.
The present invention also provides the method for preparing the L-tryptophane, and it may further comprise the steps:
A) fermentation coryneform bacterium is especially according to the Corynebacterium glutamicum of claim 1.
" open reading-frame (ORF), gene or the allelic copy of production tryptophane " is interpreted as and means, and all have the tryptophane of raising and produce open reading-frame (ORF), gene or the allelotrope of this effect after enhancing/mistake is expressed.
These comprise following open reading-frame (ORF) except that other, gene or allelotrope: aroA, aroB, aroC, aroD, aroE, aroG, aroK, cstA, eno, gap, gap2, gnd, ppsA, rpe, serA, serB, serC, tal, thyA, tkt, tpi, trpA, trpB, trpC comprises at least a A215T of being selected from (215 L-Ala replaces with Threonine) alternatively, D138A (138 aspartic acid replaces with L-Ala), the trpD that the amino acid of S149F (149 Serine replaces with phenylalanine) and A162E (162 L-Ala replaces with L-glutamic acid) is replaced, trpE comprises for example trpE of amino acid replacement S38R (38 Serine replaces with arginine) FBR, trpG comprises sudden change W14 alternatively *TrpL, zwa1 comprises that alternatively amino acid replaces the zwf of A213T (213 L-Ala replaces with Threonine).These summaries and explanation are in table 10.These especially comprise and comprise trpE, trpG, trpD, trpC and trpA and the tryptophan operon of trpL alternatively.In addition, these are also particularly including the trpE of coding colors propylhomoserin resistance o-amino benzoyl acid synthase FBRAllelotrope.
" feedback " resistance o-amino benzoyl acid synthase is interpreted as and means, compare with the wild-type form, the o-amino benzoyl acid synthase that the inhibition that is caused by tryptophane or 5-fluorotryptophan (people such as Matsui, Journal of Bacteriology169 (11): 5330-5332 (1987)) or analogue is had lower (at least 5% to 10%, 10% to 15% or 10% to 20%) susceptibility.The bacterial strain of producing the L-tryptophane comprises o-amino benzoyl acid synthase (referring to for example US 6180373 and EP0338474) such " feedback " resistance or desensitization usually.
In the 3rd or the 4th site, can integrate open reading-frame (ORF) (ORF), gene or the allelic the 3rd or the 4th copy of producing tryptophane.Except that other, following open reading-frame (ORF), gene or nucleotide sequence can be used as integration site: ccpA1, ccpA2, citA, citB, citE, cysE, gluA, gluB, gluC, gluD, glyA, luxR, luxS, lysR1, lysR2, lysR3, menE, pgi, pheA, poxB and zwa2.These summaries and explanation are in table 11.
The site of being mentioned not only comprises the open reading-frame (ORF) mentioned or the coding region of gene certainly, comprise that also being positioned at the upstream is responsible for the zone or the nucleotide sequence of expressing and regulating and control, such as the binding site and the attenuator of ribosome bind site, promotor, modulin binding site, regulation and control Yeast Nucleic Acid.These zones are usually located in coding region upstream 1-800,1-600,1-400,1-200,1-100 or 1-50 the Nucleotide scope.Equally, be positioned at the zone in downstream, in being also included within such as transcription terminator.These zones are usually located in coding region downstream 1-400,1-200,1-100,1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region, just not have the nucleotide sequence of encoding function.At last, be generally comprised within prophage in the karyomit(e) or defective phage or phage assembly and also can be used for this.
Represent the example of the Corynebacterium glutamicum chromosomal region of intergenic region, prophage, defective phage or phage assembly to be shown in table 12 and 13.The position in DNA zone is with reference to Corynebacterium glutamicum ATCC 13032 Genome Atlas shown in EP-A-1108790 or European Molecular Bioglogy Laboratory (EMBL, Heidelberg, Germany and Cambridge, the Britain) database.
Table 10
Produce open reading-frame (ORF), gene and the allelotrope of tryptophane
The gene title Coded enzyme or proteinic explanation Reference Accession number
aroA Enolpyruvylshikimate Phosphate Synthase EC 2.5.1.19 (enol pyruvic acid shikimic acid 3-phosphate synthase) People such as O ' Donohue, NCBI AF114233
aroB Dehydroquinate Synthetase EC 4.6.1.3 (dehydroquinic acid synthetic enzyme) People such as Burke, NCBI AF124600
aroC Chorismate Synthase EC 4.6.1.4 (chorismate synthase) People such as Burke, NCBI AF124600
aroD Dehydroquinate Dehydratase EC 4.2.1.10 (dehydroquinate dehydratase) People such as Joy, NCBI AF124518
aroE Shikimate Dehydrogenase People such as Joy, NCBI AF124518
EC 1.1.1.25 (shikimate dehydrogenase)
aroG Dehydro-3-Deoxyphosphoheptonate Aldolase EC4.1.2.15 (dehydrogenation-3-deoxidation phosphoric acid heptonic acid zymohexase) People such as Chen, FEMS Microbioliology Letters 107:223-230 (1993). L07603
aroK Shikimate Kinase EC 2.7.1.71 (shikimate kinase) People such as Burke, NCBI AF124600
cstA Carbon Starvation Protein A (the hungry albumin A of carbon) EP1108790 WO0100804 AX120811 AX066109
eno Enolase EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) People such as EP1108790 WO0100844 EP1090998 Hermann, Electrophoresis 19:3217-3221 (1998) AX127146 AX064945 AX136862
gap Glyceraldehyde-3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) People such as EP1108790 WO0100844 Eikmanns, Journal of Bacteriology 174:6076-6086 (1992) AX127148 AX064941 X59403
gap2 Glyceraldehyde-3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) EP1108790 WO0100844 AX127146 AX064939
gnd 6-Phosphogluconate Dehydrogenase EC 1.1.1.44 (6-Phosphogluconic dehydrogenase) EP1108790 WO0100844 AX127147 AX121689 AX065125
ppsA Phosphoenolpyruvate Synthetase Ec 2.7.9.2 (phosphoenolpyruvate synthase) EP1108790 AX127144 AX120700
rpe Ribulose-Phosphate Epimerase EC 5.1.3.1 (ribulose-phosphoric acid epimerase) EP1108790 AX127148 AX121852
serA Phosphoglycerate Dehydrogenase EC1.1.1.95 (phosphoglycerate dehydrogenase) EP1108790 AX127147 AX121499
serB Phosphoserine Phosphatase EC 3.1.3.3 EP1108790 AX127144 AX120551
(phosphoserine phosphatase)
serC Phosphoserine Aminotransferase EC 2.6.1.52 (phosphoserine aminotransferase) EP1108790 AX127145 AX121012
tal Transaldolase EC 2.2.1.2 (transaldolase) WO0104325 AX076272
thyA Thymidylate Synthase EC 2.1.1.45 (thymidylate synthase) EP1108790 AX121026 AX127145
tkt Transketolase EC 2.2.1.1 (transketolase) People such as Ikeda, NCBI AB023377
tpi Triose-phosphate Isomerase EC 5.3.1.1 (triose-phosphate isomerase) Eikmanns，Journal of Bacteriology 174：6076-6086 (1992) X59403
trpA Tryptophane Synthase (alpha Kette) EC 4.2.1.20 (tryptophan synthetase (α chain)) People such as Matsui, Nucleic Acids Research 14:10113-10114 (1986) X04960
trpB Tryptophane Synthase (β Kette) EC 4.2.1.20 (tryptophan synthetase (β chain)) People such as Matsui, Nucleic Acids Research 14:10113-10114 (1986) X04960
trpC Phosphoribosylanthranilate Isomerase EC 5.3.1.24 (phosphoric acid ribosyl o-aminobenzoic acid isomerase) People such as Matsui, Nucleic Acids Research 14:10113-10114 (1986) X04960
trpD Anthranilate Phosphoribosyltransferase EC 2.4.2.18 (anthranilic acid phosphoribosyltransferase) People such as Matsui, Nucleic Acids Research 14:10113-10114 (1986) X04960
trpD A125T， D138A， S149F， A162E The exchange of Anthranilate Phosphoribosyltransferase EC 2.4.2.18 anthranilic acid (phosphoribosyltransferase) amino acid A125T, D138A, S149F, A162E People such as O ' Gara, Applied and Environmental Microbiology 61:4477-4479 (1995)
trpE Anthranilate Synthase Komponente I EC 4.1.3.27 People such as Matsui, Nucleic Acids X04960
(o-amino benzoyl acid synthase component I) Research 14：10113-10114 (1986)
trpE fbr Anthranilat Synthase Component I feedback resistent EC 4.1.3.27 (the o-amino benzoyl acid synthase component I of feedback resistance) People such as Matsui, Journal of Bacteriology 169:5330-5332 (1987)
trpG Anthranilate Synthase Komponente II EC 4.1.3.24 (o-amino benzoyl acid synthase component I I) People such as Matsui, Nucleic Acids Research 14:10113-10114 (1986) X04960
trpL Trp Operon Leader Peptide (trp operon guiding peptide) People such as Matsui, Nucleic Acids Research 14:10113-10114 (1986) X04960
trpL W14 * Trp Operon Leaderpeptid (trp operon guiding polypeptide mutant W14 *) People such as Herry, Appliedand Environmental Microbiology 59:791-799 (1993)
zwal Cell Growth Factor 1 (somatomedin 1) EP1111062 AX133781
zwf Glucose-6-phosphat1-1-Dehydrogenase EC 1.1.1.49 (G-6-P-1-desaturase) EP1108790 WO0104325 AX127148 AX121827 AX076272
zwf A213T Glucose-6-phosphate-1-Dehydrogenase EC 1.1.1.49 (G-6-P-1-desaturase) amino acid exchange A213T EP1108790
Table 11
Be used to integrate open reading-frame (ORF), gene and the allelic target site of producing tryptophane
The gene title Coded enzyme or proteinic explanation Reference Accession number
ccpA1 Catabolite Control Protein (catabolite control albumin A 1) WO0100844 EP1108790 AX065267 AX127147
ccpA2 Catabolite Control Protein (catabolite control albumin A 2) WO0100844 EP1108790 AX065267 AX121594
citA Sensor-Kinase CitA (transmitter kinase c itA) EP1108790 AX120161
citB Transcription Regulator CitB (transcriptional regulation protein CitB) EP1108790 AX120163
citE Citrate-Lyase EC 4.1.3.6 (citrate lyase) WO0100844 EP1108790 AX065421 AX127146
cysE Serine O-Acetyltransferase EC 2.3.1.30 (Serine O-acetyltransferase) EP1108790 AX122902
gluA Glutamate Transport ATP-binding Protein (glutamate transport ATP-is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluB Glutamate-binding Protein (L-glutamic acid is conjugated protein) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluC Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
gluD Glutamate Transport Permease (L-glutamic acid transports system's permease) People such as Kronemeyer, Journal of Bacteriology 177 (5): 1152-8 (1995) X81191
glyA Glycine hydroxymethyltransferase EC 2.1.2.1 (glycine hydroxymethyltransferase) JP1997028391 E12594
luxR Transkription Regulator LuxR (transcriptional regulation protein LuxR) WO0100842 EP1108790 AX065953 AX123320
luxS Histidine Kinase LuxS (histidine kinase LuxS) EP1108790 AX123323 AX127153
lysR1 Transkription Regulator LysR1 (transcriptional regulation protein LysR1) EP1108790 AX064673 AX127144
lysR2 Transkription Activator LysR2 (transcriptional regulation protein LysR2) EP1108790 AX123312
lysR3 Transkription Regulator LysR3 (transcriptional regulation protein LysR3) WO0100842 EP1108790 AX065957 AX127150
menE O-Succinylbenzoic acid-CoA-Ligase EC 6.2.1.26 (O-succinyl-phenylformic acid-CoA ligase enzyme) WO0100843 EP1108790 AX064599 AX064193 AX127144
pgi Glucose-6-Phosphate-Isomerase EC 5.3.1.9 EP1087015 EP1108790 AX136015 AX127146
(G-6-P isomerase)
pheA Prephenate Dehydratase EC 4.2.1.51 (prephenate dehydratase) People such as Follettie, Journal of Bacteriology 167:695-702 (1986) M13774
poxB Pyruvate-Oxidase EC 1.2.3.3 (pyruvic oxidase) WO0100844 EP1096013 AX064959 AX137665
zwa2 Cell Growth Factor 2 (growth factor-2) EP1106693 EP1108790 AX113822 AX127146
The present invention correspondingly also provides preparation to produce the method for the coryneform bacterium of L-tryptophane, comprises
A) separating at least one is produced purpose ORF, gene or the allelic nucleotide sequence of tryptophane, comprise alternatively expressing and/or adjustment signal,
B) for ORF, gene or allelic 5 ' and 3 ' end the nucleotide sequence of target site is provided, wherein said site is selected from intergenic region, prophage, defective phage or phage assembly,
C) purpose ORF, gene or the allelic nucleotide sequence that preferably will have a target site nucleotide sequence is integrated into and do not duplicate in coryneform bacterium or only carry out in the limited carrier that duplicates,
D) with b) or described nucleotide sequence c) or carrier shift in the coryneform bacterium and
E) separate target site and be integrated with coryneform bacterium according to a) nucleotide sequence.The target site place do not remain in the microorganism can/make it possible to carry out episomal replication, or can/make it possible to take place swivel base, or give the nucleotide sequence of its antibiotics resistance.
Table 12
As the intergenic region of integrating open reading-frame (ORF), gene and allelic target site
Reference Accession number Sequence start position The sequence final position
EP1108790 AX120085 192176 194501
EP1108790 AX127145 235840 237311
EP1108790 AX127145 236096 237311
EP1108790 AX127148 322628 330877
EP1108790 AX127148 334045 336467
EP1108790 AX127148 289565 291841
EP1108790 AX127149 154823 161111
EP1108790 AX127149 190088 193497
EP1108790 AX127149 27398 28707
EP1108790 AX127149 61478 62944
EP1108790 AX127149 116234 117561
EP1108790 AX127149 140847 144605
EP1108790 AX127150 113274 114324
EP1108790 AX127152 244281 246403
Table 13
Be suitable for integrating open reading-frame (ORF), gene and target site allelic and coding phage or phage assembly
Reference Accession number Sequence start position The sequence final position
EP1108790 AX127149 50474 51049
EP1108790 AX127149 67886 68587
EP1108790 AX127151 72893 73480
EP1108790 AX127149 88231 89445
EP1108790 AX127148 139781 140155
EP1108790 AX127148 140546 141001
EP1108790 AX127149 194608 195294
EP1108790 AX127147 200185 200940
EP1108790 AX127147 208157 208450
EP1108790 AX127149 269616 269948
EP1108790 AX127148 336468 338324
EP1108790 AX127148 342235 342681
EP1108790 AX127148 343518 345356
EP1108790 AX127148 345872 346207
In research work of the present invention, with lysC FBRAllelic second copy is integrated into the gluB gene of Corynebacterium glutamicum, like this, the gluB gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM13994glu ∷ lysC, carries lysC in its natural lysC site FBRAllelotrope lysC T311I and in second site (target site) also is gluB gene place, carries the lysC of second copy FBRAllelotrope lysC T311I.Can help to realize with lysC FBRThe plasmid that allelotrope is integrated into the gluB gene is shown in accompanying drawing 1.It is called pK18mobsacBglu1_1.
In research work of the present invention, with the lysC of a copy FBRAllelotrope is integrated into the Corynebacterium glutamicum gluB gene as target site, like this, the gluB gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM12866glu ∷ lysC, carries the lysC gene of wild-type in its natural lysC site, and in second site (target site), also is gluB gene place, and the form of carrying is lysC FBRSecond copy lysC gene of allelotrope lysC T311I.It has been deposited in German microorganism and cell culture preservation center, and deposit number is DSM15039.Can help to realize with lysC FBRThe plasmid that allelotrope is integrated into the gluB gene is shown in accompanying drawing 1.It is called pK18mobsacBglu1_1.
In research work of the present invention, with the lysC of a copy FBRAllelotrope is integrated into the Corynebacterium glutamicum aecD gene as target site, like this, the aecD gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM12866aecD ∷ lysC, carries the lysC gene of wild-type in its natural lysC site, and in second site (target site), also is aecD gene place, and the form of carrying is lysC FBRSecond copy lysC gene of allelotrope lysC T311I.Can help to realize with lysC FBRThe plasmid that allelotrope is integrated into the aecD gene is shown in accompanying drawing 2.It is called pK18mobsacBaecD1_1.
In research work of the present invention, with the lysC of a copy FBRAllelotrope is integrated into the Corynebacterium glutamicum pck gene as target site, like this, the pck gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM12866pck ∷ lysC, carries the lysC gene of wild-type in its natural lysC site, and in second site (target site), also is pck gene place, and the form of carrying is lysC FBRSecond copy lysC gene of allelotrope lysC T311I.Can help to realize with lysC FBRThe plasmid that allelotrope is integrated into the pck gene is shown in accompanying drawing 3.It is called pK18mobsacBpck1_1.
In research work of the present invention, the ddh gene integration of a copy is advanced Corynebacterium glutamicum gluB gene as target site, like this, the gluB gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM12866glu ∷ ddh, carries the ddh gene of a copy in its natural ddh site, and in second site (target site), also is gluB gene place, carries the ddh gene of second copy.Can help to realize that the plasmid that the ddh gene integration is advanced the gluB gene is shown in accompanying drawing 4.It is called pK18mobsacBgluB2_1.
In research work of the present invention, the dapA gene integration of a copy is advanced Corynebacterium glutamicum aecD gene as target site, like this, the aecD gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM12866aecD ∷ dapA, carries the dapA gene of a copy in its natural dapA site, and in second site (target site), also is aecD gene place, carries the dapA gene of second copy.Can help to realize that the plasmid that the dapA gene integration is advanced the aecD gene is shown in accompanying drawing 5.It is called pK18mobsacBaecD2_1.
In research work of the present invention, the pyc allelotrope of a copy is integrated into Corynebacterium glutamicum pck gene as target site, like this, the pck gene locus with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of DSM12866pck ∷ pyc, carries the pyc gene of wild-type in its natural pyc site, and in second site (target site), also is pck gene place, and the form of carrying is second copy pyc gene of pyc allelotrope pyc P458S.Can help to realize that the plasmid that pyc allelotrope is integrated into the pck gene is shown in accompanying drawing 6.It is called pK18mobsacBpck1_3.
In research work of the present invention, the tryptophan operon of a copy is integrated into zone (target site) between the Corynebacterium glutamicum gene that is selected from table 12, like this, target site with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called the bacterial strain of ATCC21850nc ∷ trp, carry the tryptophan operon of a copy in its natural tryptophan operon site, it is the tryptophan operon of strains A TCC 21850, and in second site (target site), also promptly be 27398 to 28707 intergenic region place, carry the described tryptophan operon of second copy according to table 12 position.Can help to realize that the plasmid that described tryptophan operon is integrated into described target site is shown in accompanying drawing 9.It is called pK18mobsacBnc ∷ trp.
In research work of the present invention, the tryptophan operon of a copy is integrated into the Corynebacterium glutamicum zone (target site) of the coding prophage, defective phage or the phage assembly that are selected from table 12, like this, target site with regard to do not remain in the microorganism can/make it possible to carry out episomal replication nucleotide sequence, can/make it possible to the nucleotide sequence that the nucleotide sequence of swivel base takes place and give its antibiotics resistance.This is called ATCC21850pha1 ∷ trp bacterial strain, carry the tryptophan operon of a copy in its natural tryptophan operon site, it is the tryptophan operon of strains A TCC 21850, and in second site (target site), also promptly be 88231 to 89445 location, carry the described tryptophan operon of second copy according to table 13 position.Can help to realize that the plasmid that described tryptophan operon is integrated into described target site is shown in accompanying drawing 10.It is called pK18mobsacBpha1 ∷ trp.
Be to produce compound, coryneform bacterium prepared in accordance with the present invention can cultured continuously or is carried out discontinuous cultivation with batch process (batch culture) or fed-batch (feed supplement method) or repeated fed-batch method (repeating the feed supplement method).The general introduction of known cultural method is described in Chmiel (Bioprozesstechnik 1.Einf ü hrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) in the textbook).
Employed substratum must meet the requirement of specific bacterial strain in suitable mode.The explanation of relevant different microorganisms substratum has in the handbook " Manual of Methodsfor Genera Bacteriology " (Washington D.C., USA, 1981) of U.S.'s bacteriology meeting.
Such as carbohydrate as glucose, sucrose, lactose, fructose, maltose, molasses, starch and Mierocrystalline cellulose and carbohydrate, such as oils as soybean oil, Oleum Helianthi, peanut oil and Oleum Cocois and fat, such as the lipid acid as palmitinic acid, stearic acid and linolic acid, such as the alcohols as glycerol and ethanol, and, can be used as carbon source such as the organic acid as acetate or lactic acid.These materials can be used alone or as a mixture.
Nitrogen-containing organic compound, such as peptone, yeast extract, meat extract, malt extract, corn impregnation liquid, soyflour and urea, perhaps mineral compound such as ammonium sulfate, ammonium chloride, ammonium phosphate, volatile salt and ammonium nitrate, can be used as nitrogenous source.Nitrogenous source can be used alone or as a mixture.
Phosphoric acid, potassium primary phosphate or dipotassium hydrogen phosphate or corresponding sodium salts can be used as the phosphorus source.Substratum also must comprise such as sal epsom or the so necessary metal-salt of growth of ferric sulfate.At last, except that above-mentioned substance, can also use such as amino acid and the so essential growth substance of VITAMIN.In addition, can also in substratum, add suitable precursor substance.The initial substance of being mentioned can be added in the substratum by single, perhaps can add with the suitable way feed supplement in culturing process.
Basic cpd, such as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal liquor, or acidic cpd, such as phosphoric acid or sulfuric acid, the mode that can suit is used to control the pH of culture.Can use such as the such antifoams of fatty acid polyglycol ester comes control foam to form.Can in substratum, add and have the active suitable material of selection such as microbiotic, to keep the stability of plasmid.For keeping the aerobic situation, in substratum, import oxygen and oxygen-containing gas mixture, such as air.Culture temperature is generally 20 ℃ to 45 ℃, and preferred 25 ℃ to 40 ℃.Continue to cultivate purpose compound until forming maximum.Usually in 10 to 160 hours, reach this target.
Have been found that and especially produce the coryneform bacterium of L-Methionin, have unforeseeable high stability according to coryneform bacterium of the present invention.Their keep stable 10-20,20-30 at least, 30-40,40-50, preferably 50-60,60-70,70-80 and 80-90 generation or cell division cycle at least.
The preservation of following microorganism:
According to budapest treaty, bacterial strain Corynebacterium glutamicum DSM12866glu ∷ lysC is deposited in German microorganism and cell culture preservation center (DSMZ) (Braunschweig with the pure growth form on June 5th, 2002, Germany), deposit number is DSM15039.
According to budapest treaty, (=DH5alphamcr/pK18mobsacBglu1_1) pure growth form is deposited in German microorganism and cell culture preservation center April 20 calendar year 2001 to plasmid pK18mobsacBglu1_1, and deposit number is DSM14243 with coli strain DH5 α mcr/pK18mobsacBglu1_1.
According to budapest treaty, (=DH5alphamcr/pK18mobsacBaecD1_1) pure growth form is deposited in German microorganism and cell culture preservation center on June 5th, 2002 to plasmid pK18mobsacBaecD1_1, and deposit number is DSM15040 with coli strain DH5 α mcr/pK18mobsacBaecD1_1.
Embodiment 1
With lysC FBRAllelic second copy is integrated in the karyomit(e) of strain DSM 13994 and strain DSM 12866
Select to make Corynebacterium glutamicum strain DSM13994 by repeatedly non-direct mutagenesis, selection and mutant from Corynebacterium glutamicum ATCC13032.This bacterial strain has resistance to lysine analogues S-(2-amino-ethyl)-L-halfcystine, and has the insensitive feedback resistance of the inhibition E.C. 2.7.2.4. that causes by Methionin and Threonine mixture (respectively being 25mM).The lysC of this bacterial strain FBRThe allelotrope nucleotides sequence lists makes SEQ ID NO:3.It is also referred to as lysC T311I hereinafter.The proteinic aminoacid sequence of coded E.C. 2.7.2.4. shows makes SEQID NO:4.According to budapest treaty, the pure growth of this bacterial strain is preserved in German microorganism and cell culture preservation center (DSMZ) January 16 calendar year 2001.
By non-direct mutagenesis and the best mutant of selection L-Methionin accumulation, made strain DSM 12866 from Corynebacterium glutamicum ATCC13032.It is the methionine(Met) responsive type.Can on the minimum medium that contains the L-methionine(Met), regrow by adding Threonine.This bacterial strain has the wild-type lysC gene shown in SEQ ID NO:1.The proteinic aminoacid sequence of corresponding wild type E.C. 2.7.2.4. shows makes SEQ ID NO:2.According to budapest treaty, the pure growth of this bacterial strain was preserved in German microorganism and cell culture preservation center (DSMZ) on June 10th, 1999.
1.1 the lysC allelic dna of isolated strains DSM13994 and order-checking
(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strain DSM 13994 with ordinary method.Go out to have lysC gene or allelic DNA section with polymerase chain reaction (PCR) amplification.According to known Corynebacterium glutamicum lysC gene order (people such as Kalinowski, Molecular Microbiology, 5 (5), 1197-1204 (1991); Accession number X57226), select following primer tasteless nucleotide to carry out PCR:
lysC1beg(SEQ ID No：5)：
5′TA(G GAT CC)T CCG GTG TCT GAC CAC GGT G 3′
lysC2end：(SEQ ID NO：6)：
5′AC(G GAT CC)G CTG GGA AAT TGC GCT CTT CC 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A guide to Methods and Applications, 1990, Academic Press).These primers can amplify the DNA section that carries lysC gene or allelotrope, is about 1.7kb.These primers also comprise the cleavage site sequence of restriction enzyme BamHI in addition, and it uses the parenthesis mark in nucleotide sequence as implied above.
In 0.8% agarose gel electrophoresis, identify being about 1.7kb and carrying the allelic dna fragmentation of lysC of strain DSM 13994 of being amplified, and from gel, separate and purifying (QIAquick gel extraction agent box with ordinary method, Qiagen, Hilden).
With Topo TA clone's test kit (Invitrogen, Leek, The Netherlands, catalog number K4600-01) fragment is connected into carrier pCRII-TOPO then.Connector is transformed into coli strain TOP10 (Invitrogen, Leek, Holland).By with converted product contain kantlex (50mg/l), (5-bromo-4-chloro-3-indyl-β-D-galactopyranose glycosides, coated plate selects to carry the cell of plasmid to X-Gal on LB agar 64mg/l).
After the DNA isolation,, and in sepharose, identify by the plasmid that restricted cutting survey obtained.The gained plasmid is called pCRIITOPOlysC.
With PE Applied Biosystems (Weiterstadt, Germany) " ABI Prism377 " sequencing device is also determined the dna fragmentation that increased or the nucleotide sequence of PCR product according to people's such as Sanger dideoxy chain termination (Proceedings ofthe National Academy of Sciences USA, 74:5463-5467 (1977)).The coding region sequence of PCR product is shown in SEQ ID No:3.The proteinic aminoacid sequence of associated E.C. 2.7.2.4. is shown in SEQ ID NO:4.
LysC at strain DSM 13994 FBRThe 932nd of allelic coding region nucleotide sequence (SEQID NO:3) found the base thymus pyrimidine.The base cytosine(Cyt) has been found in corresponding position at wild type gene (SEQ ID NO:1).
Found the amino acid Isoleucine the 311st of the proteinic aminoacid sequence of the E.C. 2.7.2.4. of strain DSM 13994 (SEQ IDNo:4).The amino acid Threonine has been found in corresponding position at wild-type protein (SEQ ID NO:2).
Contain the base thymus pyrimidine, also be coded in the proteinic LysC allelotrope of E.C. 2.7.2.4. that the 311st of aminoacid sequence contains the amino acid Isoleucine thus the 932nd of coding region, be called lysC hereinafter FBRAllelotrope or lysC T311I.
According to budapest treaty, carry lysC FBRThe plasmid pCRIITOPOlysC of allelotrope lysC T311I is deposited in German microorganism and cell culture preservation center with the pure growth form of coli strain TOP10/pCRIITOPOlysC April 20 calendar year 2001, and deposit number is DSM14242.
1.2 make up replacement vector pK18mobsacBglu1_1
Make the chromosomal DNA donor with Corynebacterium glutamicum strain ATCC13032.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strains A TCC13032 with ordinary method.By the polymerase chain reaction, amplify the dna fragmentation that has gluB gene and neighboring area thereof.Sequence (people such as Kronemeyer according to known Corynebacterium glutamicum gluABCD gene cluster, Journal of Bacteriology, 177:1152-1158 (1995)) (accession number X81191), select following primer tasteless nucleotide to carry out PCR:gluBgl1 (SEQ ID NO:7):
5′TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3′
gluBgl2(SEQ ID NO：8)：
5′AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that carries gluB gene and neighboring area thereof, the about 1.7kb of size.The neighboring area is to be positioned at the gluB upstream, to represent gluA gene 3 ' end, to be about the sequence section of 0.33kb, and is positioned at the gluB downstream, represents 5 of gluC gene ' end, is about the sequence section of 0.44kb.These primers also comprise the cleavage site sequence of restriction enzyme BglII in addition, and it uses the parenthesis mark in nucleotide sequence as implied above.
In 0.8% agarose gel electrophoresis, identify being about 1.7kb and carrying the dna fragmentation of bacterial strain gluB gene and neighboring area thereof of being amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
With TOPO TA clone's test kit (Invitrogen, Leek, The Netherlands, catalog number K4600-01) fragment is connected into carrier pCRII-TOPO then.Connector is transformed into coli strain TOP10 (Invitrogen, Leek, Holland).By with converted product contain kantlex (50mg/l), (5-bromo-4-chloro-3-indyl-β-D-galactopyranose glycosides, coated plate selects to carry the cell of plasmid to X-Gal on LB agar 64mg/l).
After the DNA isolation,, and in sepharose, identify with the plasmid that restricted cutting survey obtained.The gained plasmid is called pCRII-TOPOglu.
With Restriction Enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) cutting plasmid pCRII-TOPOglu, and using QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolate the gluB fragment that is about 1.7kb, it is connected with the described movably cloning vector of people pK18mobsacB (Gene 14:69-73 (1994)) such as Sch_fer.This cloning vector is in advance with Restriction Enzyme BamHI cutting and with alkaline phosphatase (AlkalinePhosphatase, Boehringer Mannheim) dephosphorylation, mix with the gluB fragment that is about 1.7kb, with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle this mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBglu1.
Isolate plasmid DNA from the strain DSM 14242 (referring to embodiment 1.1) that carries plasmid pCRIITOPOlysC, and with Restriction Enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) cutting, with QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolated and be about 1.7kb, contained lysC FBRDna fragmentation, and it is linked to each other with above-mentioned carrier pK18mobsacBglu1.This cloning vector is in advance with Restriction Enzyme BamHI cutting and with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim) dephosphorylation, with the lysC that is about 1.7kb FBRFragment is mixed, and handles this mixture with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany).
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan afterwards, In:DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select the cell that carries plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBglu1_1.This plasmid map is shown in accompanying drawing 1.
According to budapest treaty, (=DH5alphamcr/pK18mobsacBglu1_1) pure growth form is deposited in German microorganism and cell culture preservation center April 20 calendar year 2001 to plasmid pK18mobsacBglu1_1, and deposit number is DSM14243 with coli strain DH5 α mcr/pK18mobsacBglu1_1.
1.3 utilize replacement vector pK18mobsacBglu1_1 with lysC FBRSecond copy of allelotrope lysCT311I is integrated into the karyomit(e) (target site: the gluB gene) of strain DSM 13994
Schedule of operation according to people such as Sch_fer (Journal of Microbiology 172:1663-1666 (1990)) is advanced Corynebacterium glutamicum strain DSM13994 with the carrier pK18mobsacBglu1_1 that describes among the embodiment 1.2 by conjugal transfer.This carrier can not be independently duplicated in DSM13994, and only just can be retained in the cell after being integrated into karyomit(e).With joiner the LB agar that has added 15mg/l kantlex and 50mg/l nalidixic acid (people such as Sambrook, Molecular Cloning:A Laboratory Manual. second edition, Cold SpringHarbor, New York, 1989) go up coated plate, select the clone or the transconjugant that have integration attitude pK18mobsacBglu1_1.The kalamycin resistance transconjugant is being contained coated plate on the LB agar of 25mg/l kantlex, and cultivating 48 hours down at 33 ℃.
In order to select will to be cloned in the wherein cut mutant of plasmid in the LB liquid nutrient medium and to cultivate 20 hours owing to the generation recombination event second time makes, containing coated plate on the LB agar of 10% sucrose then, cultivated 48 hours.
Plasmid pK18mobsacBglu1_1 as initial plasmid pK18mobsacB, except that comprising kalamycin resistance gene, also comprises the sacB gene of coding subtilis (Bacillus subtilis) levansucrase of a copy.This can be caused forming levansucrase by the expression of sucrose induction, and this enzyme catalysis is synthetic to the virose product Polylevulosan of Corynebacterium glutamicum.Therefore have only those because the clone that the second time, recombination event was excised the pK18mobsacBglu1_1 that is wherein integrated just can grow on LB agar.According to the position that the second time, recombination event took place, after excision, lysC FBRAllelic second gluB seat that copies out in the present karyomit(e) perhaps keeps host's original gluB seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of about 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.At this, amplified a dna fragmentation that carries gluB gene and neighboring area thereof from these clones' chromosomal DNA.Selected to carry out PCR with the described identical primer tasteless nucleotide that is used to make up integrated plasmid of embodiment 1.2.
gluBgl1(SEQ ID NO：7)：
5′TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3′
gluBgl2(SEQ ID NO：8)：
5′AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3′
These primers can amplify the dna fragmentation that size is about 1.7kb in having the contrast clone at original gluB seat.Have the lysC of second copy at karyomit(e) gluB seat from those FBRAmong allelic those clones, amplified the big or small dna fragmentation that is about 3.4kb.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in the same way, it also contains the lysC of second copy at karyomit(e) gluB seat except a copy that contains at the lysC seat FRBAllelotrope lysCT311I.This clone is called strain DSM 13994glu ∷ lysC.
1.4 utilize replacement vector pK18mobsacBglu1_1 with lysC FBRThe second copy lysC gene integration of this form of allelotrope lysCT311I advances the karyomit(e) (target site: the gluB gene) of strain DSM 12866
Described in embodiment 1.3, Corynebacterium glutamicum strain DSM12866 is advanced in plasmid pK18mobsacBglu1_1 conjugal transfer.Identified a clone in the mode described in the embodiment 1.3, it also contains lysC at karyomit(e) gluB seat except the lysC seat contains the wild type gene of a copy FBRThe second copy lysC gene of this form of allelotrope lysC T311I.This clone is called strain DSM 12866glu ∷ lysC.
According to budapest treaty, contain the lysC of second copy at gluB gene place FBRAllelic Corynebacterium glutamicum strain of the present invention is deposited in German microorganism and cell culture preservation center with the pure growth form of Corynebacterium glutamicum strain DSM12866glu ∷ lysC on June 5th, 2002, and deposit number is DSM15039.
1.5 make up replacement vector pK18mobsacBpck1_1
Make the chromosomal DNA donor with Corynebacterium glutamicum strain ATCC13032.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strains A TCC13032 with ordinary method.Use the polymerase chain reaction, amplify the dna fragmentation that has pck gene and neighboring area thereof.According to known Corynebacterium glutamicum pck gene order (EP1094111, with people such as Riedel, Journal of Molecular andMicrobiological Biotechnology 3:573-583 (2001)) (accession number AJ269506), select following primer tasteless nucleotide to carry out PCR:
pck_beg(SEQ ID NO：9)：
5′TA(A GAT CT)G CCG GCA TGA CTT CAG TTT 3′
pck_end(SEQ ID NO：10)：
5′AC(A GAT CT)G GTG GGA GCC TTT CTT GTT ATT 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guideto Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that carries pck gene and adjacent domain thereof, the about 2.9kb of size.These primers also comprise the cleavage site sequence of restriction enzyme BglII, use the parenthesis mark in nucleotide sequence as implied above.
In 0.8% agarose gel electrophoresis, identify being about 2.9kb and carrying the dna fragmentation of pck gene and neighboring area thereof of being amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
With TOPO TA clone's test kit (Invitrogen, Leek, The Netherlands, catalog number K4600-01) fragment is connected into carrier pCRII-TOPO then.Connector is transformed into coli strain TOP10 (Invitrogen, Leek, Holland).Converted product coated plate on the LB agar that contains kantlex (50mg/l), X-Gal (64mg/l) is selected to carry the cell of plasmid.
After the DNA isolation,, and in sepharose, identify with the plasmid that restricted cutting survey obtained.The gained plasmid is called pCRII-TOPOpck.
With Restriction Enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) cutting plasmid pCRII-TOPOpck, and using QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolate the pck fragment that is about 2.9kb, it is connected with the described movably cloning vector of people pK18mobsacB (Gene 14:69-73 (1994)) such as Sch_fer.This cloning vector is in advance with Restriction Enzyme BamHI cutting and with alkaline phosphatase (AlkalinePhosphatase, Boehringer Mannheim) dephosphorylation, mix with the pck fragment that is about 2.9kb, with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle this mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBpck1.
Isolate plasmid DNA from the strain DSM 14242 (referring to embodiment 1.1) that carries plasmid pCRIITOPOlysC, and with Restriction Enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) cutting, with QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolated and be about 1.7kb, contained lysC FBRDna fragmentation, and it is linked to each other with above-mentioned carrier pK18mobsacBpck1.This cloning vector is in advance with Restriction Enzyme BamHI cutting and with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim) dephosphorylation, with the lysC that is about 1.7kb FBRFragment is mixed, and handles this mixture with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany).
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan afterwards, In:DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select the cell that carries plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobdsacBpck1_1.This plasmid map is shown in accompanying drawing 3.
1.6 utilize replacement vector pK18mobsacBpck1_1 the second copy lysC gene integration of this form of lysCFBR allelotrope lysCT311I to be advanced the karyomit(e) (target site: the pck gene) of strain DSM 12866
As described in embodiment 1.3, Corynebacterium glutamicum strain DSM12866 is advanced in the plasmid pK18mobsacBpck1_1 conjugal transfer described in the embodiment 1.5.As described in embodiment 1.3, select the target recombination event in the Corynebacterium glutamicum DSM12866 karyomit(e).According to the position that the second time, recombination event took place, after excision, lysC FBRAllelic second pck seat that copies out in the present karyomit(e) perhaps keeps host's original pck seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of about 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.At this, amplified a dna fragmentation that carries pck gene and neighboring area thereof from these clones' chromosomal DNA.Selected to carry out PCR with the described identical primer tasteless nucleotide that is used to make up integrated plasmid of embodiment 1.5.
pck_beg(SEQ ID NO：9)：
5′TA(A GAT CT)G CCG GCA TGA CTT CAG TTT 3′
pck_end(SEQ ID NO：10)：
5′AC(A GAT CT)G GTG GGA GCC TTT CTT GTT ATT 3′
These primers can amplify the dna fragmentation that size is about 2.9kb in having the contrast clone at original pck seat.Have the lysC of second copy at karyomit(e) pck seat from those FBRAmong allelic those clones, amplified the big or small dna fragmentation that is about 4.6kb.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it also has lysC at karyomit(e) pck seat except have the wild type gene copy at the lysC seat FBRThe second copy lysC gene of this form of allelotrope lysC T311I.This clone is called strain DSM 12866pck::lysC.
1.7 make up replacement vector pK18mobsacBaecD1_1
Make the chromosomal DNA donor with Corynebacterium glutamicum strain ATCC13032.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strains A TCC13032 with ordinary method.By the polymerase chain reaction, amplify the dna fragmentation that has aceD gene and neighboring area thereof.According to known Corynebacterium glutamicum aecD gene order (people such as Rossol, Journal of Bacteriology 174:2968-2977 (1992)) (accession number M89931), select following primer tasteless nucleotide to carry out PCR:
aecD_beg(SEQ ID NO：11)：
5′GAA CTT ACG CCA AGC TGT TC 3′
aecD_end(SEQ ID NO：12)：
5′AGC ACC ACA ATC AAC GTG AG 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that carries aecD gene and neighboring area thereof, the about 2.1kb of size.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is about 2.1kb that is amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
Dna fragmentation with Restriction Enzyme BamHI and EcoRV (Amersham Pharmacia, Freiburg, Germany) cutting purifying.Afterwards this fragment is connected into carrier pUC18 (people such as Norrander, Gene 26:101-106 (1983)).This carrier is in advance with Restriction Enzyme BglII and SmaI cutting, and dephosphorylation mixes with the fragment that is about 1.5kb, carry aecD, and usefulness T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) is handled this mixture.Connector is transformed into coli strain TOP10 (Invitrogen, Leek, The Netherlands).With conversion product coated plate on the LB agar that contains kantlex (50mg/l) and X-Gal (64mg/l), select the cell that carries plasmid thus.
After the DNA isolation, the plasmid that obtains is checked by Restriction Enzyme cutting, identifies in sepharose.The gained plasmid is called pUC18aecD.
Isolate plasmid DNA from the strain DSM 14242 (referring to embodiment 1.1) that carries plasmid pCRIITOPOlysC, and with Restriction Enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) cutting and afterwards with the processing of Klenow polysaccharase.After separating in sepharose (0.8%) with QIAquick gel extraction agent box (Qiagen, Hilden, Germany), from sepharose, isolate and be about 1.7kb, contain lysC FBRDna fragmentation, and it is connected with above-mentioned carrier pUC18aecD.This carrier is in advance with Restriction Enzyme StuI cutting and with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) dephosphorylation, with the lysC that is about 1.7kb FBRFragment is mixed, and handles this mixture with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany).
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan afterwards, In:DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select the cell that carries plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pUC18aeCD1.
Plasmid pUC18 aecD1 handles with the Klenow polysaccharase afterwards with Restriction Enzyme KpnI cutting.Use Restriction Enzyme SalI (Amersham-Pharmacia then, Freiburg, Germany) cut this plasmid, by QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolate the fragment that carries aecD and lysC, is about 3.2kb, and it be connected with the described movably cloning vector pK18mobsacB of people (Gene14:69-73 (1994)) such as Sch_fer.This carrier is in advance with Restriction Enzyme SmaI and SalI cutting, and with alkaline phosphatase (AlkalinePhosphatase, Boehringer Mannheim) dephosphorylation, mix with the fragment that is about 3.2kb, have aecD and a lysC, and with T4 dna ligase (Amer sham-Pharmacia, Freiburg, Germany) handle this mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBaecDl_1.This plasmid map is shown in accompanying drawing 2.
According to budapest treaty, (=DH5alphamcr/pK18mobsacBaecD1_1) pure growth form is deposited in German microorganism and cell culture preservation center on June 5th, 2002 to plasmid pK18mobsacBaecD1_1, and deposit number is DSM15040 with coli strain DH5 α mcr/pK18mobsacBaecD1_1.
1.8 utilize the replacement vector pK18mobsacBaecD1-1 will be as lysC FBRThe allelic second copy lysC gene integration advances the karyomit(e) (target site: the aecD gene) of strain DSM 12866
As described in embodiment 1.3, Corynebacterium glutamicum strain DSM12866 is advanced in the plasmid pK18mobsacBaecD1_1 conjugal transfer described in the embodiment 1.4.As described in embodiment 1.3, select the target recombination event in the Corynebacterium glutamicum DSM12866 karyomit(e).According to the position that the second time, recombination event took place, after excision, lysC FBRAllelic second aecD seat that copies out in the present karyomit(e) perhaps keeps host's original aecD seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of about 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.At this, from these clones' chromosomal DNA, amplified a dna fragmentation that carries aecD gene and neighboring area thereof.Selected to carry out PCR with the described identical primer tasteless nucleotide that is used to make up integrated plasmid of embodiment 1.7.
aecD_beg(SEQ ID NO：11)：
5′GAA CTT ACG CCA AGC TGT TC 3′
aecD_end(SEQ ID NO：12)：
5′AGC ACC ACA ATC AAC GTG AG 3′
These primers can amplify the dna fragmentation that size is about 2.1kb in having the contrast clone at original aecD seat.Have the second copy lysC at karyomit(e) aecD seat from those FBRAmong the allelic clone, amplify the dna fragmentation that size is about 3.8kb.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it also has lysC at karyomit(e) aecD seat except have the wild type gene copy at the lysC seat FBRThe second copy lysC gene of this form of allelotrope lysC T311I.This clone is called strain DSM 12866aecD ∷ lysC.
Embodiment 2
The ddh gene integration of second copy is advanced the karyomit(e) (target site: the gluB gene) of strain DSM 12866
2.1 make up replacement vector pK18mobsacBglu2_1
Make the chromosomal DNA donor with Corynebacterium glutamicum strain ATCC13032.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strains A TCC13032 with ordinary method.By the polymerase chain reaction, amplify the dna fragmentation that has gluB gene and neighboring area thereof.According to known Corynebacterium glutamicum gluABCD gene cluster sequence (people such as Kronemeyer, Journal of Bacteriology, 177:1152-1158 (1995); EP1108790) (accession number X81191 and AX127149), select following primer tasteless nucleotide to carry out PCR:
gluA_beg(SEQ ID NO：13)：
5′CAC GGT TGC TCA TTG TAT CC 3′
gluD_end(SEQ ID NO：14)：
5′CGA GGC GAA TCA GAC TTC TT 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that size is about 4.4kb, carries gluB gene and neighboring area thereof.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
With TOPO TA clone's test kit (Invitrogen, Leek, The Netherlands, catalog number K4600-01) this fragment is connected into carrier pCRII-TOPO.Connector is transformed into coli strain TOP10 (Invitrogen, Leek, Holland).With conversion product coated plate on the LB agar that contains kantlex (50mg/l) and X-Gal (64mg/l), select the cell that carries plasmid thus.
After the DNA isolation, the plasmid that obtains is checked by Restriction Enzyme cutting, identifies in sepharose.The gained plasmid is called pCRII-TOPOglu2.
Plasmid pCRII-TOPOglu2 Restriction Enzyme EcoRI and SalI (Amersham-Pharmacia, Freiburg, Germany) cutting, with QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolate the gluB fragment that is about 3.7kb, and it is linked to each other with the described movably cloning vector pK18mobsacB of people (Gene 14,69-73 (1994)) such as Sch_fer.This carrier is in advance with Restriction Enzyme EcoRI and SalI cutting and with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim) dephosphorylation, mix with the gluB fragment that is about 3.7kb, and with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle this mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBglu2.
Described in embodiment 2.1, utilize the polymerase chain reaction also to amplify one section dna fragmentation that carries the ddh gene.According to known Corynebacterium glutamicum ddh gene cluster sequence (people such as Ishino, Nucleic Acids Research 15,3917 (1987)) (accession number Y00151),
Select following primer tasteless nucleotide to carry out PCR:
ddh_beg(SEQ ID NO：15)：
5′CTG AAT CAA AGG CGG ACA TG 3′
ddh_end(SEQ ID NO：16)：
5′TCG AGC TAA ATT AGA CGT CG 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that size is about 1.6kb, has the ddh gene.
In 0.8% agarose gel electrophoresis, identify being about 1.6kb and carrying the dna fragmentation of ddh gene of being amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
Behind the purifying, the fragment that carries the ddh gene is connected with above-mentioned carrier pK18mobsacBglu2.This carrier has been done partly cutting with Restriction Enzyme BamHI in advance.By using Klenow polysaccharase (Amersham-Pharmacia, Freiburg, Germany) handle carrier, outstanding being put down by benefit of incision tip is blunt end, then this carrier is mixed with the dna fragmentation that is about 1.6kb, have a ddh gene, and with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) treating mixture.By Vent polysaccharase (New England Biolabs, Frankfurt, Germany) being used for the PCR reaction, having produced to have blunt end and being suitable for connecting into through dna fragmentation among the pretreated carrier pK18mobsacBglu2, band ddh.
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan afterwards, In:DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select the cell that carries plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBglu2_1.This plasmid map is shown in accompanying drawing 4.
2.2 utilize replacement vector pK18mobsacBglu2_1 the ddh gene integration of second copy to be advanced the karyomit(e) (target site: the gluB gene) of strain DSM 12866
As described in embodiment 1.3, Corynebacterium glutamicum strain DSM12866 is advanced in the plasmid pK18mobsacBglu2_1 conjugal transfer described in the embodiment 2.1.As described in embodiment 1.3, select the target recombination event in the Corynebacterium glutamicum DSM12866 karyomit(e).According to the position that the second time, recombination event took place, after excision, the ddh gene of second copy appears at the gluB seat in the karyomit(e), perhaps keeps host's original gluB seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of about 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.At this, amplified a dna fragmentation that carries described glu zone from these clones' chromosomal DNA.Selected and be used to make up the identical primer tasteless nucleotide that replaces plasmid described in the embodiment 2.1 and carry out PCR.
gluA_beg(SEQ ID NO：13)：
5′CAC GGT TGC TCA TTG TAT CC 3′
gluD_end(SEQ ID NO：14)：
5′CGA GGC GAA TCA GAC TTC TT 3′
These primers can amplify the dna fragmentation that size is about 4.4kb in having the contrast clone at original glu seat.From the clone who has the second copy ddh gene at karyomit(e) glu seat, amplify the dna fragmentation that size is about 6kb.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it also has the second copy ddh gene except having at the ddh seat the copy at karyomit(e) gluB seat.This clone is called strain DSM 12866glu ∷ ddh.
Embodiment 3
The dapA gene integration of second copy is advanced the karyomit(e) (target site: the aecD gene) of strain DSM 12866
3.1 make up replacement vector pK18mobsacBaecD2_1
Make the chromosomal DNA donor with Corynebacterium glutamicum strain ATCC13032.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strains A TCC13032 with ordinary method.By the polymerase chain reaction, amplify the dna fragmentation that has aecD gene and neighboring area thereof.According to known Corynebacterium glutamicum aecD gene order (people such as Rossol, Journal of Bacteriology 174:2968-2977 (1992)) (accession number M89931), select following primer tasteless nucleotide to carry out PCR:
aecD_beg(SEQ ID NO：11)：
5′GAA CTT ACG CCA AGC TGT TC 3′
aecD_end(SEQ ID NO：12)：
5′AGC ACC ACA ATC AAC GTG AG 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that carries aecD gene and adjacent domain thereof, the about 2.1kb of size.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is about 2.1kb that is amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
Dna fragmentation with Restriction Enzyme BglII and EcoRV (Amersham Pharmacia, Freiburg, Germany) cutting purifying.Afterwards this fragment is connected into carrier pUC18 (people such as Norrander, Gene 26:101-106 (1983)).This carrier is in advance with Restriction Enzyme BamHI and SmaI cutting, and dephosphorylation mixes with the fragment that is about 1.5kb, carry aecD, and usefulness T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) is handled this mixture.Connector is transformed into coli strain TOP10 (Invitrogen, Leek, The Netherlands).With conversion product coated plate on the LB agar that contains kantlex (50mg/l) and X-Gal (64mg/l), select the cell that carries plasmid thus.
After the DNA isolation, the plasmid that obtains is checked by Restriction Enzyme cutting, identifies in sepharose.The gained plasmid is called pUC18aecD.
Utilize the polymerase chain reaction, amplify the dna fragmentation that another has dapA gene and neighboring area thereof.According to known Corynebacterium glutamicum dapA gene order (people such as Bonassi, Nucleic Acids Research 18:5421 (1990)) (accession number X53993 and AX127149), select following primer tasteless nucleotide to carry out PCR:
dapA_beg(SEQ ID NO：17)：
5′CGA GCC AGT GAA CAT GCA GA 3′
dapA_end(SEQ ID NO：18)：
5′CTT GAG CAC CTT GCG CAG CA 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that carries dapA gene and adjacent domain thereof, the about 1.4kb of size.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is about 1.4kb that is amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
Behind the purifying, the dna fragmentation that is about 1.4kb, carries dapA is connected with above-mentioned carrier pUC18aecD.This carrier with Restriction Enzyme StuI cutting, mixes with the dna fragmentation that is about 1.4kb in advance, and with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) treating mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan afterwards, In:DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select the cell that carries plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pUC18aecD2.
Plasmid pUC18 aecD2 partly cuts (Amersham-Pharmacia with Restriction Enzyme SalI cutting and with EcoRI, Freiburg, Germany), utilize QIAquick gel extraction agent box (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, from sepharose, isolate the fragment that is about 2.7kb, has aecD and dapA, and it be connected with the described movably cloning vector pK18mobsacB of people (Gene 14:69-73 (1994)) such as Sch_fer.This cloning vector is in advance with Restriction Enzyme EcoRI and SalI cutting and with alkaline phosphatase (Alkaline Phosphatase, BoehringerMannheim) dephosphorylation, mix with the fragment that has aecD and dapA, be about 2.7kb, with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle this mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBaecD2_1.Plasmid map is shown in accompanying drawing 5.
3.2 the second dapA gene integration that copies is advanced (target site: the aecD gene) in strain DSM 12866 karyomit(e)s with replacement vector pK18mobsacBaecD2_1
As described in embodiment 1.3, Corynebacterium glutamicum strain DSM12866 is advanced in the plasmid pK18mobsacBaecD2_1 conjugal transfer described in the embodiment 3.1.As described in embodiment 1.3, select the target recombination event in the Corynebacterium glutamicum DSM12866 karyomit(e).According to the position that the second time, recombination event took place, after excision, the dapA gene of second copy appears at the aecD seat in the karyomit(e), perhaps keeps host's original aecD seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of about 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.At this, from these clones' chromosomal DNA, amplified a dna fragmentation that carries aecD gene and neighboring area thereof.Selected to carry out PCR with the identical primer tasteless nucleotide that is used to make up integrated plasmid described in the embodiment 3.1.
aecD_beg(SEQ ID NO：11)：
5′GAA CTT ACG CCA AGC TGT TC 3′
aecD_end(SEQ ID NO：12)：
5′AGC ACC ACA ATC AAC GTG AG 3′
These primers can amplify the dna fragmentation that size is about 2.1kb in having the contrast clone at original aecD seat.Have the clone of the second copy dapA gene at karyomit(e) aecD seat from those, amplify the dna fragmentation that size is about 3.6kb.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it also has the dapA gene of second copy except having at the dapA seat the copy at chromosomal aecD seat.This clone is called strain DSM 12866aecD ∷ dapA.
Embodiment 4
The second copy pyc gene integration of this form of pyc allelotrope pycP458S is advanced the karyomit(e) (target site: the pck gene) of strain DSM 12866
4.1 make up replacement vector pK18mobsacBpck1_3
With the replacement vector pK18mobsacBpck1 described in the embodiment 1.5 as the allelic carrier is carrier of slotting pyc.
As described in embodiment 2.1, also amplify the dna fragmentation that has pyc gene and neighboring area thereof with the polymerase chain reaction.According to known Corynebacterium glutamicum pyc gene cluster sequence (people such as Peters-Wendisch, Journal of Microbiology 144:915-927 (1998)) (accession number Y 09548), select following primer tasteless nucleotide to carry out PCR:
pyc_beg(SEQ ID NO：19)：
5′TC(A CGC GT)C TTG AAG TCG TGC AGG TCA G 3′
pyc_end(SEQ ID NO：20)：
5′TC(A CGC GT)C GCC TCC TCC ATG AGG AAG A 3′
Shown in primer synthetic by MWG Biotech, and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press).These primers can amplify the dna fragmentation that size is about 3.6kb, has the pyc gene.These primers also comprise the cleavage site sequence of restriction enzyme MluI, and it uses the parenthesis mark in nucleotide sequence as implied above.
Be about 3.6kb, have the dna fragmentation of pyc gene with what restriction enzyme MluI cutting amplified, identify by the electrophoresis in 0.8% sepharose, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
Behind the purifying, the fragment that will have the pyc gene is connected into described carrier pK18mobsacBpckl.This carrier is in advance with Restriction Enzyme BssHII cutting, with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) dephosphorylation, mix with the dna fragmentation that has pyc gene, about 3.6kb, and with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) treating mixture.
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan afterwards, In:DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select the cell that carries plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBpck1_2.
4.2 make up pyc allelotrope pyc P458S by wild-type pyc gene being carried out site-specific mutagenesis
(Stratagene, La Jolla USA) finish the site directed mutagenesis with the site-directed mutagenesis kit of QuikChange.EP-A-1108790 has described can improve the point mutation that L-Methionin is produced in the Corynebacterium glutamicum pyc gene.Become the point mutation of thymus pyrimidine based on the 1372nd cytosine(Cyt) of pyc gene nucleotide series, thereby cause in the 458th proline(Pro) to be replaced by Serine by this nucleotide sequence deutero-aminoacid sequence.This equipotential gene is called pyc P458S.For forming described sudden change, select following primer tasteless nucleotide to be used for linear amplification: P458S-1 (SEQ ID NO:21):
5′GGATTCATTGCCGATCAC(TCG) CACCTCCTTCAGGCTCCA 3′
P458S-2(SEQ ID NO：22)：
5′GTGGAGGAAGTCCGAGGT(CGA) GTGATCGGCAATGAATCC 3′
Shown in primer synthetic by MWG Biotech.The codon of Serine that is used for replacing 458 proline(Pro) is at nucleotide sequence as implied above parenthesis mark.The primer that plasmid pK18mobsacBpck1_2 described in the embodiment 4.1 and two are complementary to a chain of this plasmid separately is used to utilize the linear amplification of Pfu Turbo archaeal dna polymerase.Prolong by this primer, formed and be the mutant plasmid that disconnects loop chain.Handle the linear amplification product with DpnI (this restriction endonuclease specificity cutting methylates and hemimethylated template DNA).Carrier DNA this new synthetic, disconnection, sudden change is transformed into coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)).After the conversion, the fracture (break) in this mutant plasmid of XLl Blue cytothesis.Select transformant containing on the LB substratum of kantlex 50mg/l.After the DNA isolation, utilize restricted cutting to check the plasmid that is obtained, in sepharose, identify.Check the segmental dna sequence dna of mutant DNA by order-checking.The sequence of PCR product is consistent with described sequences of people (2002) such as Ohnishi.The gained plasmid is called pK18mobsacBpck1_3.This plasmid map is shown in accompanying drawing 6.
4.3 the second copy pyc gene integration of this form of pyc allelotrope pycP458S is advanced the karyomit(e) (target site pck gene) of strain DSM 12866 with replacement vector pkl8mobsacBpck1_3
As described in embodiment 1.3, Corynebacterium glutamicum strain DSM12866 is advanced in the plasmid pK18mobsacBpck1_3 conjugal transfer described in the embodiment 4.2.As described in embodiment 1.3, select the target recombination event in the Corynebacterium glutamicum DSM12866 karyomit(e).According to the position that the second time, recombination event took place, after excision, the pyc allelotrope of second copy appears at the pck seat in the karyomit(e), perhaps keeps host's original pck seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of about 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.At this, from these clones' chromosomal DNA, amplified a dna fragmentation that carries pck gene and neighboring area thereof.Selected and be used to make up the identical primer tasteless nucleotide that replaces plasmid described in the embodiment 1.5 and carry out PCR.
pck_beg(SEQ ID NO：9)：
5′TA(A GAT CT)G CCG GCA TGA CTT CAG TTT 3′
pck_end(SEQ ID NO：10)：
5′AC(A GAT CT)G GTG GGA GCC TTT CTT GTT ATT 3′
These primers can amplify the dna fragmentation that size is about 2.9kb in having the contrast clone at original pck seat.From have the allelic clone of the second copy pyc at karyomit(e) pck seat, amplify the dna fragmentation of the about 6.5kb of size.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it also has the second copy pyc gene of this form of pyc allelotrope pycP458S except the pyc seat has the wild type gene of a copy at karyomit(e) pck seat.This clone is called strain DSM 12866pck ∷ pyc.
Embodiment 5
Preparation Methionin
Corynebacterium glutamicum strain DSM13994glu ∷ lysC, DSM12866glu ∷ lysC, DSM12866pck ∷ lysC, DSM12866aecD ∷ lysC, DSM12866glu ∷ ddh, DSM12866aecD ∷ dapA and the DSM12866pck ∷ pyc that obtains in embodiment 1,2,3 and 4 cultivated being suitable for produce in the nutritional medium of Methionin, and measure lysine content in the culture supernatants.
For this reason, at first culture is gone up cultivation 24 hours at brain-heart agar plate (Merck, Darmstadt, Germany) at 33 ℃.From this agar plate culture, inoculate pre-culture (containing the 10ml substratum in the 100ml Erlenmeyer flask).With the substratum of MM substratum as pre-culture.On the shaking table of 240rpm, cultivated pre-culture 24 hours in 33 ℃.From this pre-culture inoculation master culture, the initial OD (660nm) that makes master culture is 0.1OD.Also the MM substratum is used for master culture.
The MM substratum
CSL 5g/l
MOPS 20g/l
Glucose (high pressure heat sterilization separately) 50g/l
Salt:
(NH 4) 2SO 4 25g/l
KH 2PO 4 0.1g/l
MgSO 4*7H 2O 1.0g/l
CaCl 2*2H 2O 10mg/l
FeSO 4*7H 2O 10mg/l
MnSO 4*H 2O 5.0mg/l
Vitamin H (sterile filtration) 0.3mg/l
VitB1 * HCl (sterile filtration) 0.2mg/l
CaCO 3 25g/l
With ammoniacal liquor CSL (corn impregnation liquid), MOPS (morpholino propane sulfonic acid) and salts solution are transferred to pH7, and the high pressure heat sterilization.Add aseptic substrate and vitamin solution then, and with the CaCO of drying regime high pressure heat sterilization 3
In containing the 100ml band baffle plate Erlenmeyer flask of 10ml volume substratum, finish cultivation.In 33 ℃ and 80% atmospheric moisture, cultivate.
After 48 hours, (Beckmann Instruments GmbH Munich) measures the wavelength place at 660nm and measures OD with Biomek 1000.Amino acidanalyser with Eppendorf-BioTronik (Hamburg, Germany) is measured formed lysine amount by ion exchange chromatography and post-column derivation triketohydrindene hydrate detection method.
Experimental result is shown in table 14.
Table 14
Bacterial strain OD (660nm) Methionin HCl g/l
DSM13994 12.0 19.1
DSM13994glu∷lysC 9.9 20.0
DSM12866 12.5 14.9
DSM15039 11.4 16.2
DSM12866pck∷lysC 12.6 16.5
DSM12866aecD∷lysC 12.0 15.9
DSM12866glu∷ddh 11.0 15.5
DSM12866aecD∷dapA 11.1 16.2
DSM12866pck∷pyc 10.9 16.9
Embodiment 6
The lysC_T3111I-allelotrope of a copy is integrated into strain DSM 13992 chromosomal intergenic region ncode1
6.1 make up exchange carrier pK18mobsacBnc1 ∷ lysC
Make the chromosomal DNA donor with Corynebacterium glutamicum strain DSM13994 (referring to embodiment 1).(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from strain DSM 13994 with ordinary method.By polymerase chain reaction (PCR), amplify the dna fragmentation (SEQ ID NO:23) in zone between chromogene with mark work " ncode1 ".This zone is positioned at the 27398th to 28707 of Corynebacterium glutamicum gene group sequence, and it can obtain from login code AX127149 (referring to table 12).According to this regional known array, select following primer tasteless nucleotide to carry out PCR:
Primer ncode 1 (SEQ ID NO:24):
5′GA(A GAT CT)A AGC TCT ATT GTC CCC TAC G 3′
Primer ncode 2 (SEQ ID NO:25):
5′ GAT CCT TTT AAA AGC CAG TAA CAA G 3′
Primer ncode 3 (SEQ ID NO:26):
5′CTT GTT ACT GGC TTT TAA AA G GAT CCT_ATT AAA GAA CAC TCC CCTAT 3′
Shown in primer synthetic by MWG Biotech company, and according to people's such as Innis standard pcr (PCR protocols.A guide to methods and applications, 1990, Academic Press) carries out PCR, wherein at first amplifying two kinds of products with combination of primers ncode 1 and ncode 2 or ncode 3 and ncode 4, is that template and combination of primers ncode 1 and ncode 4 one are used from PCR for the second time with these two kinds of products then.In this mode, selected primer can amplify the dna fragmentation of the about 1.2kb of size, and this fragment has the interface (emphasizing (SEQID NO:28) with underscore) of a synthetical restriction enzyme BarnHI at intergenic region.In addition, these primers also comprise the interface sequence of restriction enzyme Bglll, and it indicates with bracket in above-mentioned Nucleotide series.
In 0.8% agarose gel electrophoresis, identify being about 1.2kb, having the dna fragmentation of intergenic region ncode1 of being amplified, and with ordinary method from gel, separate and purifying (QIAquick gel extraction agent box, Qiagen, Hilden).
Next, with Topo TA clone's test kit (Invitrogen, Leek, Netherlands, catalog number K4600-01) this fragment is connected into carrier pCRII-TOPO.With connector transform into coli strain TOP10 (Invitrogen, Leek, Netherlands).With conversion product coated plate on the LB agar that contains kantlex (50mg/l) and X-Gal (64mg/l), select the cell that carries plasmid thus.
After the DNA isolation, the gained plasmid is confirmed by restricted fracture and is identified in sepharose.The gained plasmid is called pCRII-TOPOnc.
With Restriction Enzyme BglII (Amersham-Pharmarcia, Freiburg, Germany) cutting plasmid pCRII-TOPOnc, and in sepharose (0.8%), separate subsequently, with Qiagenquick gel extraction agent box (Qiagen, Hilden, Germany) from sepharose, isolate the fragment that is about 1.2kb, this fragment is connected with described movably cloning vector pK18mobsacB of people (Gene 14:69-73 (1994)) such as Sch_fer.This carrier at first disconnects with Restriction Enzyme BarnHI and with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim) dephosphorylation, mix with the fragment that is about 1.2kb, and with T4-DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle this culture.
Afterwards, with connecting culture transformed into escherichia coli bacterial strain DH5 α (people such as Grant; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA cloning.A practical approach. the 1st volume .ILR-Press, Cold Spring Harbor, New York, 1989).Be added with the LB agar of 25mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual with transforming culture.Second edition, Cold SpringHarbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen company is isolated plasmid DNA from transformant, and by carry out with enzyme Xbal restricted cutting and subsequently agarose gel electrophoresis check.This plasmid is called pK18mobsacBnc.
With the plasmid pCRIITOPOlysC described in Restriction Enzyme BamHI (Amersham-Pharmarcia, Freiburg, Germany) the cutting embodiment 1.With Qiagenquick gel extraction agent box (Qiagen, Hilden, Germany) after separating in sepharose (0.8%), from sepharose, isolate and be about 1.7kb, comprise lysC TThe dna fragmentation of 311I, and it is connected with above-mentioned carrier pK18mobsacBnc.This carrier at first disconnects with Restriction Enzyme BamHI and with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) dephosphorylation, mix with the fragment that is about 1.7kb, and with T4-DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle this culture.
Afterwards, with connecting culture transformed into escherichia coli bacterial strain DH5omcr (LifeTechnologies GmbH, Karlsruhe, Germany) (Hanahan, In.DNA cloning.A practical approach. the 1st volume .ILR-Press, Cold Spring Harbor, New York, 1989).Be added with the LB agar of 25mg/l kantlex (people such as Sambrook, Molecular Cloning:A laboratory manual with transforming culture.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.QIAprep Spin Miniprep test kit with Qiagen company is isolated plasmid DNA from transformant, and by carrying out restricted cutting with enzyme HindIII and Xbal and agarose gel electrophoresis is subsequently checked it.This plasmid is called pK18mobsacBnc ∷ lysC.Accompanying drawing 7 has shown the collection of illustrative plates of this plasmid.
6.2 utilize exchange carrier pK18mobsacBnc ∷ lysC with IYS CFBThe second copy lysC gene integration of this form of M allelotrope lysCT311I advances the karyomit(e) (target site: intergenic region ncode1) of strain DSM 13992
Evolutionary approach (1990Journal of Microbiology172:1663-1666) according to people such as Sch_fer shifts into Corynebacterium glutamicum strain DSM13992 with the carrier pK18mobsacBnc ∷ lysC that names among the embodiment 6.1.
Select to prepare Corynebacterium glutamicum strain DSM13992 by the mutagenesis of multiple non-directional, selection and mutant from Corynebacterium glutamicum ATCC13032.This strains strepto-have resistance and lysine analogues S-(2-amino-ethyl)-L-halfcystine is had resistance on phenotype.Yet this bacterial strain has the wild-type E.C. 2.7.2.4. of the inhibition sensitivity that the mixture by Methionin and Threonine (each 25mM) is caused.According to budapest treaty, the pure growth of this bacterial strain was registered in German microorganism and cell culture preservation center (DSMZ, Braunschweig, Germany) January 16 calendar year 2001.
Carrier pK18mobsacBnc ∷ lysC can not be independently duplicated in DSM13992, and only just can be retained in the cell after being integrated into karyomit(e) by reorganization.
To engage culture the LB agar that has added 15mg/l kantlex and 50mg/l nalidixic acid (people such as Sambrock, Molecular Cloning:A Laboratory Manual. second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select the clone that those have the pK18mobsacBnc ∷ lysC of integration thus.With the coated plate on the LB agar plate that contains the 25mg/l kantlex that is cloned in that begins to grow, cultivated 16 hours in 33 ℃.In order to realize in the LB liquid nutrient medium, cultivating after 16 hours with initial these clones of 10% sucrose by recombination event excision second time plasmid.Plasmid pK18mobsacB comprises the sacB gene of a copy, and this gene can be to the virose Polylevulosan of Corynebacterium glutamicum with sucrose inversion.
Therefore, have only clone that the pK18mobsacBnc ∷ lysC of integration excised once more to grow containing on the LB agar of sucrose.The position of recombination event generation for the second time during according to excision, perhaps the lysC of a copy and peripheral intergenic region ncode1 thereof and this plasmid are together cut, perhaps only are that intergenic region ncode1 is cut.
In order to prove that the lysC copy is retained among the chromosomal intergenic region ncode1, standard pcr (PCR Protocols.A Guide to Methods andApplications according to people such as Innis, 1990, Academic Press), utilize the polymerase chain reaction to study the clone of the phenotype of about 20 performances " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing ".At this, amplified a dna fragmentation that carries lysC gene and neighboring area thereof from these clones' chromosomal DNA.Select following primer tasteless nucleotide to carry out PCR:
3371V(SEQ ID NO：29)：
5′TAT CAT GCG GTG AGC TGT GA 3′
3372N(SEQ ID NO：30)：
5′TAG GGG TGA TGT GCT ACT GT 3′
These primers can amplify the dna fragmentation that size is about 1.3kb in having the contrast clone in original ncode1 site.Among chromosomal intergenic region ncode1, have among the clone of lysC gene of a copy from those, can amplify the dna fragmentation of the about 3.0kb of size.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it also has the lysCFBR allelotrope lysC T311I of second copy except having in the lysC site the copy in chromosomal intergenic region ncode1.This clone strain DSM 13992nc ∷ lysC by name.
Embodiment 7
The ilvD gene integration of second copy is advanced the karyomit(e) (target site: the ddh gene) of brevibacterium (Brevibacteriumlactofermentum) bacterial strain FERM BP-1763
With the gene ilvD of the second coding dihydroxyacid dehydratase that copies, import the ddh gene of the coding diaminopimelate dehydrogenase of brevibacterium bacterial strain FERM BP-1763, to improve the Xie Ansuan production of this bacterial strain.
Brevibacterium bacterial strain FERM BP-1763 is the Xie Ansuan producer (US-A-5,188,948) who mycophenolic acid is had resistance.It is that L-Isoleucine and L-methionine(Met) are auxotrophic.
7.1. the DNA of the ilvD gene of isolated strains FERM BP-1763
(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) isolates chromosomal DNA from brevibacterium bacterial strain FERM BP-1763 with ordinary method.Utilize the polymerase chain reaction, amplify the DNA section that comprises the ilvD gene.The ilvD gene order of Corynebacterium glutamicum is known (Genbank accession number AJ012293; The SEQ.ID.No.1 of DE19907567, the SEQ.ID.No.7063 of accession number AX034412 and EP1108790 and 1399, accession number AX127147 and AX121483).Even existing great mass of data show brevibacterium and Corynebacterium glutamicum inequality also be very relevant (people such as Abe, Journal of General and Applied Microbiology 13:279-301 (1967); People such as Suzuki, International Journal of SystematicBacteriology 31:131-138 (1981)).Therefore, according to known Corynebacterium glutamicum ilvD gene order, select following primer tasteless nucleotide to carry out PCR:
ilvD-A1(SEQ ID NO：31)：
5′ATC GTC TCT AGA CTT GGA GCG CCA AAA GGC AC 3′
ilvD-E1(SEQ ID NO：32)：
5′GTC ATC TCT AGA TCG AGG CAG AGT TCG CTG GT 3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany), and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods andApplications, 1990, Academic Press).The dna fragmentation that these primers can increase and be about 2970bp, have ilvD gene and neighboring area thereof.These neighboring areas comprise the upstream region of the about 284bp in ilvD genes encoding zone and the downstream area of about 819bp.These primers also comprise the cleavage site sequence of restriction enzyme XbaI, and it uses the underscore mark in above-mentioned nucleotide sequence.
Cut the dna fragmentation that carries ilvD gene and upstream and downstream zone thereof that is amplified with restriction enzyme XbaI, and in 0.8% sepharose, identify by agarose gel electrophoresis.From gel, separate and be purified into the dna fragmentation (High Pure PCR Product Purification Kit, Roche DiagnosticsGmbH, Mannheim, Germany) that is about 2.95kb with ordinary method.
7.2 make up replacement vector pK18mobsacBddh ∷ ilvD
To be template from the isolating chromosomal DNA of bacterial strain FERM BP-1763 among the embodiment 7.1, to make up ddh and integrate box.The DNA section that term " ddh integrates box " expression mainly is made up of the ddh gene order, it comprises a synthetical restriction enzyme sites, can insert other gene or sequence in this site.This final structure can be used for gene or other dna sequence dna are integrated in the karyomit(e) ddh gene.
According to known Corynebacterium glutamicum ddh gene order (people such as Ishino, NucleicAcids Research 15 (9), 319 reach following (1987), Genbank accession number Y00151, and SEQ ID No.7068 and the Nr.3494 of EP1108790, accession number AX127152 and AX123578), select following primer tasteless nucleotide, utilize gene SOEing method (GeneSplicing by Overlap Extension, Horton, MolecularBiotechnology 3:93-98 (1995)), produce ddh by polymerase chain reaction (PCR) and integrate box:
ddh_del1(SEQ ID NO：33)：
5′ATC GTC GCT AGC CCA ACG AAT CTC CAC GAG ACG 3′
ddh_del2(SEQ ID NO：34)：
5′GAC ATC CGC ACC ATG CCT GA 3′
ddh_del3(SEQ ID NO：35)：
5′TCA GGC ATG GTG CGG ATG TCT AGA GTC GGC AAC CAC GAA GCA TT3′
ddh_del4(SEQ ID NO：36)：
5′ATG CTC GCT AGC ATG ACC AAC ATC CGC GTA GC 3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany), and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods andApplications, 1990, Academic Press).Primer ddh_del1 and ddh_del4 respectively comprise the cleavage site sequence (sequence of representing with underscore) of restriction enzyme NheI in above-mentioned nucleotide sequence.Primer ddh_del3 is made of two nucleotides sequence column regions, and the 653rd to 634 Nucleotide of one of them and ddh genes encoding zone combines, and another combines with the 606th to 587 Nucleotide.Between these two zones, deleted 27 base pairs in ddh genes encoding zone, and between these two zones of primer ddh_del3, inserted four bases " TAGA " to form the artificial cleavage site of a restriction enzyme XbaI.These four bases mark with tilted letter, and this XbaI site marks with underscore in above-mentioned sequence.5 of primer ddh_del3 '-stub area reverse complemental is in primer ddh_del2.
Pass through PCR, primer ddh_del1 and ddh_del2 can amplify the dna fragmentation that is about 0.75kb, it comprise 3 of ddh gene '-stub area and downstream area, primer ddh_del3 and ddh_del4 can amplify the dna fragmentation that is about 0.64kb, it comprise 5 of ddh gene '-stub area.In 0.8% sepharose, carry out agarose gel electrophoresis afterwards and check amplified production, with ordinary method (High Pure PCR ProductPurification Kit, Roche Diagnostics GmbH, Mannheim, Germany) it is separated and purifying from gel.These two amplified productions can use the template of the PCR reaction of primer ddh_del1 and ddh_del4 together as another.This produces the about 1.38kb of size, comprises the ddh integration box of ddh gene, and it comprises the disappearance of 31bp and the zone of synthetical Restriction Enzyme XbaI cleavage site and ddh gene downstream 412bp.
Cut this ddh with restriction enzyme NheI and integrate box, and identify by the agarose gel electrophoresis in 0.8% sepharose.With ordinary method (High Pure PCR ProductPurification Kit, Roche Diagnostics GmbH, Mannheim, Germany) from gel, separate and be purified into the dna fragmentation that is about 1.36kb, and it is connected with the described movably cloning vector pK18mobsacB of people (Gene145:69-73 (1994)) such as Sch_fer.This carrier pK18mobsacB is in advance with restriction enzyme XbaI cutting and with alkaline phosphatase (Boehringer, Mannheim) dephosphorylation is integrated box with ddh afterwards and is mixed, and with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle.The sticky end of restricted nickase NheI and XbaI is a complementary, and such connection product no longer can be by any cutting in these two kinds of enzymes.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBddh_Xba.
With Restriction Enzyme XbaI cut vector pK18mobsacBddh_Xba, this enzyme only cuts once in the artificial XbaI-site of ddh integration box inside.Make (Boehringer Mannheim, Germany) this carrier dephosphorylation with alkaline phosphatase then, and with it with available from embodiment 7.1, be about 2.95kb, the ilvD fragment of having cut with Restriction Enzyme XbaI mixes.(Amersham Pharmacia, Freiburg, Germany) handles this mixture with the T4DNA ligase enzyme.
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan then, In.DNA Cloning.A PracticalApproach. the 1st volume .ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold SpringHarbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen company is isolated plasmid DNA from transformant, and by restricted cutting and agarose gel electrophoresis subsequently it is checked.This plasmid is called pK18mobs acBddh ∷ ilvD.Accompanying drawing 8 has shown the collection of illustrative plates of this plasmid.
7.3 the ilvD gene integration of second copy is advanced karyomit(e) (target site: the ddh gene)
Scheme (Journal of Microbiology 172:1663-1666 (1990)) according to people such as Sch_fer is advanced brevibacterium bacterial strain FERM BP-1763 with the carrier pK18mobsacBddh ∷ ilvD conjugal transfer described in the embodiment 7.2.This carrier can not be independently duplicated in FERMBP-1763, and only just can be retained in the cell after being integrated into karyomit(e), or be integrated into ddh zone as necessary herein, or be integrated into the ilvD zone.At first, with joiner the LB agar that has added 15mg/l kantlex and 50mg/l nalidixic acid (people such as Sambrook, Molecular Cloning:A Laboratory Manual. second edition, Cold Spring Harbor, New York, 1989) go up coated plate, selected at the dyeing interbody fusion clone or the transconjugant of pK18mobsacBddh ∷ ilvD.The kalamycin resistance transconjugant is being contained coated plate on the LB agar of 25mg/l kantlex, and cultivating 48 hours down at 33 ℃.Afterwards with regard to the integration screening gained transconjugant of plasmid at the ddh seat.
Utilize polymerase chain reaction (PCR), detected the integration of the plasmid pK18mobsacBddh ∷ ilvD of 20 kalamycin resistance transconjugants at the ddh seat.The chromosomal DNA that separates these clones with ordinary method (people such as Eikmanns, Microbiology 140:1817-1828 (1994)).Select following primer tasteless nucleotide to carry out PCR:
ddh-int1(SEQ ID NO：37)：
5′-attcttccgcgaccgcgata-3′
ddh-A1(SEQ ID NO：38)：
5′-ccggataaaccaccatgaac-3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany).They are positioned at ddh integrates outside the box, can go out the PCR fragment of about 1.45kb from the clone's that is integrated with carrier pK18mobsacBddh ∷ ilvD at the ilvD seat DNA cloning.Enter the ddh seat if this size is about the vector integration of 10kb, then can not obtain the visible amplified production.Standard pcr (PCR Protocols.A Guide to Methods andApplications, 1990, Academic Press) according to people such as Innis carries out PCR.
Utilize agarose gel electrophoresis to identify the dna fragmentation that is increased.
Utilize two clones that do not show visible pcr amplification product to select wherein to make the clone that plasmid is cut owing to the generation of recombination event for the second time.To be cloned in the liquid LB substratum and cultivate 20 hours, and contain coated plate on the LB agar of 10% sucrose afterwards, and cultivating 48 hours.
As initial plasmid pK18mobsacB, plasmid pK18mobsacBddh ∷ ilvD also comprises the sacB gene of the coding subtilis levansucrase of a copy except that comprising kalamycin resistance gene.This can be caused forming levansucrase by the expression of sucrose induction, and this enzyme catalysis is synthetic to the virose product Polylevulosan of Corynebacterium glutamicum.Therefore have only those since for the second time the recombination event clone that excised the pK18mobsacBddh ∷ ilvD of integration wherein just can grow having added on the LB agar of sucrose.According to the position that the second time, recombination event took place, after excision, the ilvD gene of second copy appears at the ddh seat, perhaps keeps host's original ddh seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.(people such as Eikmanns, Microbiology140:1817-1828 (1994)) goes out chromosomal DNA from these clone and separate with ordinary method.
Select the primer tasteless nucleotide ddh-int1 described in the present embodiment and ddh-A1 (SEQ ID NO:38 and SEQ ID NO:39) and according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press) finishes PCR.These primers can amplify the dna fragmentation that comprises the ddh gene.In having the contrast clone at original ddh seat, can amplify the dna fragmentation of the about 1.65kb of size.From the clone who is integrated with the second copy ilvD gene at karyomit(e) ddh seat, can amplify the dna fragmentation of the about 4.6kb of size.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it is integrated with the ilvD gene of a copy at karyomit(e) ddh seat.This clone is called bacterial strain FERM BP-1763ddh ∷ ilvD.
Embodiment 8
The production of L-Xie Ansuan
In being suitable for producing the nutritional medium of Xie Ansuan, cultivate the brevibacterium bacterial strain FERM BP-1763ddh ∷ ilvD that obtains among the embodiment 7.3 and detect valine content in the culture supernatants.
For this reason, at first went up the cultivation bacterial strain 24 hours at agar plate (brain/heart agar) in 33 ℃.From this agar plate culture, inoculate pre-culture (containing the 10ml substratum in the 100ml Erlenmeyer flask).With complete culture medium C gIII (2.5g/l NaCl, 10g/l microbial culture peptone, 10g/l microbial culture yeast extract, pH7.4,20g/l glucose (separately high pressure heat sterilization) is as the substratum of this pre-culture.Cultivated 48 hours in 33 ℃ the shaking table of pre-culture at 240rpm.From this pre-culture inoculation master culture, (OD 660nm) is 0.1OD to make the initial optical density(OD) of master culture.Master culture uses the MM substratum.
The MM substratum
CSL (corn impregnation liquid) 5g/l
MOPS (morpholino propane sulfonic acid) 20g/l
Glucose (high pressure heat sterilization separately) 50g/l
Salt:
(NH 4) 2SO 4 25g/l
KH 2PO 4 0.1g/l
MgSO 4*7H 2O 1.0g/l
CaCl 2*2H 2O 10mg/l
FeSO 4*7H 2O 10mg/l
MnSO 4*H 2O 5.0mg/l
Isoleucine (sterile filtration) 0.1g/l
Methionine(Met) (sterile filtration) 0.1g/l
VitB1 * HCl (sterile filtration) 0.2mg/l
Leucine (sterile filtration) 0.1g/l
CaCO 3 25g/l
With ammoniacal liquor CSL, MOPS and salts solution are transferred to pH7, and the high pressure heat sterilization.Add aseptic substrate and vitamin solution then, and with the CaCO of drying regime high pressure heat sterilization 3
In the Erlenmeyer flask of the 100ml band turbulence generator (flow spoilers) that contains 10ml volume substratum, finish cultivation.In 33 ℃ and 80% atmospheric moisture, cultivate.
After 48 hours, (Beckmann Instruments GmbH Munich) measures the wavelength place at 660nm and measures OD with Biomek 1000.Amino acidanalyser with Eppendorf-BioTronik (Hamburg, Germany) is measured formed Xie Ansuan amount by ion exchange chromatography and post-column derivation triketohydrindene hydrate detection method.Experimental result is shown in table 15.
Table 15
Bacterial strain OD(660) Xie Ansuan g/l
FERM BP-1763 8.6 12.1
FERM BP-1763ddh∷ilvD 8.5 12.9
Embodiment 9
The tryptophan operon of second copy is integrated into Corynebacterium glutamicum strain ATCC21850 karyomit(e) (target site: intergenic region ncode1) (US 3,849,251)
The tryptophan operon of second copy is imported the intergenic region ncode1 (regional ncode1 is corresponding to the sequence of Corynebacterium glutamicum gene group the 27398th and 28707 interdigits, referring to table 12, embodiment 6.1 and SEQ ID NO:23) of Corynebacterium glutamicum strain ATCC21850 to improve the L-tryptophane production of this bacterial strain.
The tryptophan operon of Corynebacterium glutamicum contains gene trpL, its coding is for the very important tryptophan operon guiding peptide of decay control, gene trpE, its coding o-amino benzoyl acid synthase composition I, gene trpG, its coding o-amino benzoyl acid synthase composition II, gene trpD, its coding anthranilic acid phosphoribosyltransferase, gene trpC, its phosphoric acid ribosyl o-aminobenzoic acid isomerase of encoding, gene trpB, its coding tryptophan synthetase (β chain) and gene trpA, its tryptophan synthetase (α chain) of encoding.
Corynebacterium glutamicum strain ATCC21850 (US 3,849,251) is the tryptophane producer that the chemical analog to L-tryptophane and other die aromatischen Aminosaeuren has multiple resistance.It is that L-phenylalanine and L-tyrosine are auxotrophic.
Be listed in the 42nd from the nucleotides sequence of the gene trpL of ATCC21850 and replace guanine by VITAMIN B4.This causes encoding and guides the codon tgg of peptide the 14th amino acids tryptophane to become terminator codon tga.The position of this nonsense mutation prevents the formation of termination structure and causes the anti-termination of composing type to reply (Heery and Dunican, Applied and EnvironmentalMicrobiology 59 (3): 791-799 (1993)).
9.1. separate the DNA of the tryptophan operon that comprises strains A TCC21850
Separate chromosomal DNA with ordinary method people such as (, Microbiology 140:1817-1828 (1994)) Eikmanns from Corynebacterium glutamicum strain ATCC21850.Use the polymerase chain reaction, amplify the DNA section that has tryptophan operon.The karyomit(e) nucleotide sequence of Corynebacterium glutamicum is known, can be at patent application EP-A-1108790 and the (EMBL of European Molecular Bioglogy Laboratory, Heidelberg, Germany and Cambridge UK) find among the accession number AX114121 of nucleotide sequence database.Can also (Bethesda, MD find it in the database of NCBI USA) (NCBI) at National Library of Medicine.The accession number of the gene nucleotide series in the tryptophan operon is AX123436 (trpE), AX123438 (trpG), AX123439 (trpD), AX123440 (trpC), AX123442 (trpB) and AX123443 (trpA).Can under accession number M17892, find the nucleotide sequence of trpL.
According to known Corynebacterium glutamicum tryptophan operon sequence, select following primer tasteless nucleotide to carry out PCR:
trp-A1(SEQ ID NO：39)：
5′GTAC GGA TCC CAA GGA CAG CTC GTG TAG AC 3′
trp-E1(SEQ ID NO：40)：
5′AGCT GGA TCC CAC CGG CTC GAA GCT AAG AT 3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany), finish the PCR reaction with the TripleMaster PCR system of Eppendorf (Hamburg, Germany).These primers can amplify the dna fragmentation that is about 7.95kb, has tryptophan operon and neighboring area thereof.These primers also comprise the cleavage site sequence of restriction enzyme BamHI, and it uses the underscore mark in above-mentioned nucleotide sequence.
Cut the dna fragmentation that has tryptophan operon and upstream and downstream zone thereof that is amplified with restriction enzyme BamHI, and identify by in 0.8% sepharose, carrying out agarose gel electrophoresis.From gel, separate and purifying is about the dna fragmentation (high purity PCR product purification test kit, Roche Diagnostics GmbH, Mannheim, Germany) of 7.93kb with ordinary method.
9.2 make up replacement vector pK18mobsacBnc ∷ trp
The plasmid pK18mobsacBnc that obtains in embodiment 6.1 comprises intergenic region ncodel (SEQ ID NO:23).This plasmid is used alkaline phosphatase (Boehringer Mannheim, Germany) dephosphorylation then with Restriction Enzyme BamHI cutting, with comprise available from embodiment 9.1 tryptophan operon, with Restriction Enzyme BamHI cutting also the dna fragmentation of purifying mix.(Amersham Pharmacia, Freiburg, Germany) handles this mixture with the T4DNA ligase enzyme.
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan then, In.DNA Cloning.A PracticalApproach. the 1st volume .ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, ColdSpring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen company is isolated plasmid DNA from transformant, and by restricted cutting and agarose gel electrophoresis subsequently it is checked.This plasmid is called pK18mobsacBnc ∷ trp.The collection of illustrative plates of this plasmid is shown in accompanying drawing 9.
9.3 the tryptophan operon of second copy is integrated into regional ncode1 between chromogene
Scheme (Journal of Microbiology 172:1663-1666 (1990)) with people such as Sch_fer is advanced strains A TCC21850 with the carrier pK18mobsacBnc ∷ trp conjugal transfer described in the embodiment 9.2.This carrier can not be independently duplicated in ATCC21850, and only just can be retained in the cell after being integrated into karyomit(e), or be integrated into ncode1 zone as necessary herein, or be integrated into the tryptophan operon zone.At first, with joiner the LB agar that has added 15mg/l kantlex and 50mg/l nalidixic acid (people such as Sambrook, Molecular Cloning:A Laboratory Manual. second edition, Cold SpringHarbor, New York, 1989) go up coated plate, selected at the dyeing interbody fusion clone or the transconjugant of pK18mobsacBnc ∷ trp.The kalamycin resistance transconjugant is being contained coated plate on the LB agar of 25mg/l kantlex, and cultivating 48 hours down at 33 ℃.Just occur in the transconjugant of the integration incident screening gained in the ncode1 zone then.
Utilize polymerase chain reaction (PCR), detected the integration of the plasmid pK18mobsacBnc ∷ trp of 20 kalamycin resistance transconjugants at the ncode1 seat.The chromosomal DNA that separates these clones with ordinary method (people such as Eikmanns, Microbiology 140:1817-1828 (1994)).Select following primer tasteless nucleotide to carry out PCR:
ncode1-A1(SEQ ID NO：41)：
5′-ACT ATC ATG CGG TGA GCT GT-3′
ncodel-E1(SEQ ID NO：42)：
5′-GGC AGC TCT GTA GCC ATA TT-3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany).They are positioned at outside the cloned segment in karyomit(e) ncodel zone, can go out the PCR fragment of about 1.47kb from the DNA cloning of clone with the carrier pK18mobsacBnc ∷ trp that is integrated into tryptophan operon.If this size is entered the ncode1 zone for the vector integration of 14.9kb, will visible amplified production can not appear.Standard pcr (PCR Protocols.AGuide to Methods and Applications, 1990, Academic Press) according to people such as Innis carries out PCR.
Utilize agarose gel electrophoresis to identify the dna fragmentation that is increased.
Utilize two clones that do not show visible pcr amplification product to select wherein to make the clone that plasmid is cut owing to the generation of recombination event for the second time.To be cloned in the liquid LB substratum and cultivate 20 hours, and contain coated plate on the LB agar of 10% sucrose afterwards, and cultivating 48 hours.
As initial plasmid pK18mobsacB, plasmid pK18mobsacBnc ∷ trp also comprises the sacB gene of the coding subtilis levansucrase of a copy except that comprising kalamycin resistance gene.This can be caused forming levansucrase by the expression of sucrose induction, and this enzyme catalysis is synthetic to the virose product Polylevulosan of Corynebacterium glutamicum.Therefore have only those since for the second time the recombination event clone that excised the pK18mobsacBnc ∷ trp of integration wherein just can grow having added on the LB agar of sucrose.According to the position that the second time, recombination event took place, after excision, the tryptophan operon of second copy appears at the ncode1 seat, perhaps keeps host's original ncode1 seat.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) goes out chromosomal DNA from these clone and separate with ordinary method.
Select the primer tasteless nucleotide ncode1-A1 (SEQ IDNO:41) and another primer trp-test that describe in the present embodiment to carry out PCR:
trp-test(SEQ ID NO：43)：
5′-CCA ACC ACG ATG AGA AGG AC -3′
Standard pcr (PCR Protocols.A Guide toMethods and Applications, 1990, Academic Press) according to people such as Innis is finished PCR.These primers are about the dna fragmentation of 0.92kb only with amplification size from the clone of the trp operon that is integrated with second copy in karyomit(e) ncode1 zone.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone who between chromogene, is integrated with a copy tryptophan operon in the regional ncode1 in this way.This clone is called strains A TCC21850nc ∷ trp.
Embodiment 10
Second copy of tryptophan operon is integrated into Corynebacterium glutamicum strain ATCC21850 karyomit(e) (target site: phage assembly pha1) (US 3,849,251)
In the present embodiment, the tryptophan operon of second copy is imported the phage assembly phal (Genebank accession number AX127149: 88231-89445 position Nucleotide, referring to table 13) of Corynebacterium glutamicum ATCC21850 to improve the tryptophane production of bacterial strain.
10.1 make up replacement vector pK18mobsacBpha1 ∷ trp
Integrate the template of box as amplification phage assembly pha1 with strain separated ATCC21850 chromosomal DNA among the embodiment 9.1.The DNA section that term " phage assembly pha1 integrates box " expression mainly is made up of phage assembly pha1 sequence comprises the artificial constructed Restriction Enzyme BamHI site that can insert other gene or sequence.This final structure can be used for gene or other dna sequence dna are integrated into karyomit(e) phage assembly pha1.
Utilize polymerase chain reaction (PCR) amplification phage assembly phal to integrate box by SOEing method (Gene Splicing by Overlap Extension, Horton, Molecular Biotechnology 3:93-98 (1995)).Mentioned the nucleotide sequence of the phage assembly phal of Corynebacterium glutamicum in the table 13, it can find in the nucleotide sequence 88231-89445 position under the Genebank accession number AX127149.Select following primer to carry out PCR:
pha1-int1(SEQ ID NO：44)：
5′A GTC GTC GAC GCT ATC AAC GCT TCG ATC AC 3′
pha1-int2(SEQ ID NO：45)：
5′CTG TCT CGT ACG GAT GAC AG 3′
pha1-int3(SEQ ID NO：46)：
5′CTG TCA TCC GTA CGA GAC AG G GAT CCA TAC CTT GCC AGT CAA ATCT 3′
pha1-int4(SEQ ID NO：47)：
5′G TCA GAA TTC GCA GCA TGT AAC GGA TAG GT 3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany), and carry out PCR according to people's such as Innis standard pcr (PCR Protocols.A Guide to Methods andApplications, 1990, Academic Press).Primer pha1-int1 and pha1-int 4 comprise the cleavage site sequence of restriction enzyme SalI and EcoRI, and they indicate with underscore in above-mentioned nucleotide sequence.Primer pha1-int3 is made up of two zones of phage assembly pha1 nucleotide sequence, has deleted 17 bases of this nucleotide sequence and integrated five bases " GATCC " to form a synthetical restriction enzyme BamHI cleavage site between these two zones.These five bases indicate with tilted letter in above-mentioned sequence, and the BamHI site indicates with underscore.5 of primer pha1-int3 '-stub area reverse complemental is in primer pha1-int2.
By PCR, primer pha1-int1 and pha1-int2 can amplify the dna fragmentation that is about 0.59kb, and primer pha1-int3 and pha1-int4 can amplify the dna fragmentation that is about 0.9kb.In 0.8% sepharose, carry out agarose gel electrophoresis afterwards and check amplified production, with ordinary method (High Pure PCR Product Purification Kit, Roche Diagnostics GmbH, Mannheim, Germany) it is separated and purifying from gel.These two amplified productions can use the template of the PCR reaction of primer pha1-int1 and pha1-int4 together as another.This produces the about 1.48kb of size, comprises the phage assembly pha1 integration box of phage assembly pha1 and neighboring area thereof, and it comprises disappearance and a synthetical Restriction Enzyme BamHI cleavage site of 17bp.
Integrate box with restriction enzyme SalI and EcoRI cutting phage assembly pha1, and in 0.8% sepharose, carry out agarose gel electrophoresis and identify.With ordinary method (HighPure PCR Product Purification Kit, Roche Diagnostics GmbH, Mannheim, Germany) from gel, separate and be purified into the dna fragmentation that is about 1.47kb, and it is connected with the described movably cloning vector pK18mobsacB of people (Gene 145:69-73 (1994)) such as Sch_fer.This carrier pK18mobsacB with restriction enzyme SalI and EcoRI cutting, integrates box with phage assembly pha1 afterwards and mixes, and handle with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany) in advance.
Use connector transformed into escherichia coli bacterial strain DH5 α (people such as Grant afterwards; Proceedingsof the National Academy of Sciences USA, 87 (1990) 4645-4649) (Hanahan, In.DNA Cloning.A Practical Approach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold Spring Harbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep SpinMiniprep test kit with Qiagen is isolated plasmid DNA from transformant, and checks by restricted cutting and agarose gel electrophoresis subsequently.This plasmid is called pK18mobsacBphal.
With Restriction Enzyme BamHI cut vector pK18mobsacBpha1, this enzyme only cuts once in the artificial BamHI-site of phage assembly pha1 integration box inside.Use alkaline phosphatase (Boehringer Mannheim, Germany) to make this carrier dephosphorylation then, and with it with available from embodiment 9.1, comprise tryptophan operon, the dna fragmentation crossed with Restriction Enzyme BamHI cutting and purifying mixes.(Amersham Pharmacia, Freiburg, Germany) handles this mixture with the T4DNA ligase enzyme.
Use connector transformed into escherichia coli bacterial strain DH5 α mcr (Life TechnologiesGmbH, Karlsruhe, Germany) (Hanahan then, In.DNA Cloning.A PracticalApproach. the 1st volume, ILR-Press, Cold Spring Harbor, New York, 1989).Conversion product is being added with the LB agar of 50mg/l kantlex (people such as Sambrook, Molecular Cloning:A Laboratory Manual.Second edition, Cold SpringHarbor, New York, 1989) go up coated plate, select to carry the cell of plasmid thus.
QIAprep Spin Miniprep test kit with Qiagen company is isolated plasmid DNA from transformant, and by restricted cutting and agarose gel electrophoresis subsequently it is checked.This plasmid is called pK18mobsacBpha1 ∷ trp.Accompanying drawing 10 has shown the collection of illustrative plates of this plasmid.
10.3 second copy of tryptophan operon is integrated into karyomit(e) (target site: phage assembly pha1)
Scheme (Journal of Microbiology 172:1663-1666 (1990)) with people such as Sch_fer is advanced Corynebacterium glutamicum ATCC21850 with the carrier pK18mobsacBpha1 ∷ trp conjugal transfer described in the embodiment 10.2.This carrier can not be independently duplicated in ATCC21850, and only just can be retained in the cell after being integrated into karyomit(e), or be integrated into phage assembly pha1 as necessary herein, or be integrated into tryptophan operon.At first, with joiner the LB agar that has added 15mg/l kantlex and 50mg/l nalidixic acid (people such as Sambrook, Molecular Cloning:A LaboratoryManual. second edition, Cold Spring Harbor, New York, 1989) go up coated plate, selected at the dyeing interbody fusion clone or the transconjugant of pK18mobsacpha1 ∷ trp.The kalamycin resistance transconjugant is being contained coated plate on the LB agar of 25mg/l kantlex, and cultivating 48 hours down at 33 ℃.The gained transconjugant is screened in the integration of plasmid in phage assembly pha1 afterwards.
Utilize polymerase chain reaction (PCR), detected the integration of the plasmid pK18mobsacBpha1 ∷ trp of 20 kalamycin resistance transconjugants at phage assembly pha1.The chromosomal DNA that separates these clones with ordinary method (people such as Eikmanns, Microbiology 140:1817-1828 (1994)).Select following primer tasteless nucleotide to carry out PCR:
pha1-test1(SEQ ID NO：48)：
5′-GCG CTC CAT CAC TAG AGT TC-3′
pha1-test2(SEQ ID NO：49)：
5′-GGT CAG AGT GAG CAC CTA AG-3′
Shown in primer synthetic by MWG Biotech (Ebersberg, Germany).They are positioned at phage assembly pha1 integrates outside the box, can go out the PCR fragment of about 1.56kb from the clone's that is integrated with carrier pK18mobsacBphal ∷ trp at tryptophan operon DNA cloning.Advance phage assembly pha1 if this size is about the vector integration of 15kb, then can not obtain the visible amplified production.Standard pcr (PCR Protocols.A Guide to Methods and Applications, 1990, Academic Press) according to people such as Innis carries out PCR.
Utilize agarose gel electrophoresis to identify the dna fragmentation that is increased.
Utilize two clones that do not show visible pcr amplification product to select wherein to make the clone that plasmid is cut owing to the generation of recombination event for the second time.To be cloned in the liquid LB substratum and cultivate 20 hours, and contain coated plate on the LB agar of 10% sucrose afterwards, and cultivating 48 hours.
As initial plasmid pK18mobsacB, plasmid pK18mobsacBpha1 ∷ trp also comprises the sacB gene of the coding subtilis levansucrase of a copy except that comprising kalamycin resistance gene.This can be caused forming levansucrase by the expression of sucrose induction, and this enzyme catalysis is synthetic to the virose product Polylevulosan of Corynebacterium glutamicum.Therefore have only those because the clone that the second time, recombination event was excised the pK18mobsacBpha1 ∷ trp that is wherein integrated just can grow having added on the LB agar of sucrose.According to the position that the second time, recombination event took place, after excision, the tryptophan operon of second copy appears among the phage assembly phal, perhaps keeps host's original phage assembly pha1.
40 to 50 clones' " in growth in the presence of the sucrose " and the phenotype of " in the presence of kantlex, not growing " have approximately been detected.Investigated the clone of the phenotype of 20 demonstrations " in growth in the presence of the sucrose " and " in the presence of kantlex, not growing " with the polymerase chain reaction.(people such as Eikmanns, Microbiology 140:1817-1828 (1994)) goes out chromosomal DNA from these clone and separate with ordinary method.
Select the primer tasteless nucleotide pha1-test1 (SEQ IDNO:48) and another primer pha1-test3 that described in the present embodiment to carry out PCR:
pha1-test3(SEQ ID NO：50)：
5′-CTG CGA GCT TCA TTC GAT CC -3′
Standard pcr (PCR Protocols.A Guide toMethods and Applications, 1990, Academic Press) with people such as Innis is finished PCR.These primers are only to amplify the dna fragmentation of the about 1.6kb of size from the clone who is integrated with second tryptophan operon that copies at karyomit(e) phage assembly pha1.
In 0.8% agarose gel electrophoresis, identify the dna fragmentation that is amplified.
Identified a clone in this way, it is integrated with the tryptophan operon of a copy at pha1 place, karyomit(e) phage zone.This clone is called strains A TCC21850pha1 ∷ trp.
Embodiment 11
The production of L-tryptophane
In being suitable for producing the nutritional medium of tryptophane, cultivate available from the Corynebacterium glutamicum strain ATCC21850nc ∷ trp of embodiment 9.3 with available from the Corynebacterium glutamicum strain ATCC21850phal ∷ trp of embodiment 10.2, and detect tryptophane in the culture supernatants.
For this reason, at first went up the cultivation bacterial strain 24 hours at agar plate (brain/heart agar) in 33 ℃.From this agar plate culture, inoculate pre-culture (containing the 10ml substratum in the 100ml Erlenmeyer flask).With complete culture medium C gIII (2.5g/l NaCl, 10g/l microbial culture peptone, 10g/l microbial culture yeast extract, pH7.4,20g/l glucose (separately high pressure heat sterilization) is as the substratum of these pre-cultures.Cultivated 16 hours in 33 ℃ the shaking table of pre-culture at 240rpm.
Master culture uses the tryptophane that has added L-phenylalanine and L-tyrosine to produce substratum.
Tryptophane is produced substratum
Gravel bed 100g/l rubs
CSL (corn impregnation liquid) 10g/l
Salt:
(NH 4) 2SO 4 20g/l
KH 2PO 4 0.5g/l
MgSO 4*7H 2O 0.25g/l
K 2HPO 4 0.5mg/l
L-phenylalanine (sterile filtration) 0.3g/l
L-tyrosine (sterile filtration) 0.15g/l
Transfer to pH7 with will rub gravel bed, CSL and salts solution of ammoniacal liquor, and the high pressure heat sterilization.Add aseptic substrate then.
For the 50ml tryptophane in the 500ml Erlenmeyer flask of band turbulence generator (flow spoilers) is produced culture medium inoculated, (Beckmann Instruments GmbH Munich) measures the wavelength place at 660nm and measures optical density(OD) (OD) with Biomek1000.Centrifugal afterwards pre-culture is removed supernatant liquor, each precipitation is resuspended in the 5ml tryptophane produces in the substratum.From this suspension inoculation master culture, (OD 660nm) is 0.1OD to make the initial optical density(OD) of master culture.This culture was cultivated 5 days in 33 ℃ on the shaking table of 150rpm.
After 5 days, (Beckmann Instruments GmbH Munich) measures the optical density(OD) (OD) that the wavelength place measures each culture at 660nm with Biomek 1000.Amino acidanalyser with Eppendorf-BioTronik (Hamburg, Germany) is measured formed tryptophane amount by ion exchange chromatography and post-column derivation triketohydrindene hydrate detection method.
Experimental result is shown in table 16.
Table 16
Bacterial strain OD(660) Tryptophane HCl g/l
ATCC21850 10.3 1.0
ATCC21850nc∷trp 10.1 1.4
ATCC21850pha1∷trp 10.2 1.5
The accompanying drawing summary:
Described base pair numbering is the approximation that obtains under the situation of replicate measurement.
Accompanying drawing 1: the collection of illustrative plates of plasmid pK18mobsacBglu1_1
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
HindIII: the cleavage site of Restriction Enzyme HindIII
BamHI: the cleavage site of Restriction Enzyme BamHI
LysC:lysC FBRAllelotrope, lysC T311I
3 ' terminal fragment of ' gluA:gluA gene
GluB ': 5 ' terminal fragment of gluB gene
3 ' terminal fragment of ' gluB:gluB gene
GluC ': 5 ' terminal fragment of gluC gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 2: the collection of illustrative plates of plasmid pK18mobsacBaecD1_1
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
SalI: the cleavage site of Restriction Enzyme SalI
LysC:lysC FBRAllelotrope, lysC T311I
AecD ': 5 ' terminal fragment of aecD gene
3 ' terminal fragment of ' aecD:aecD gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 3: the collection of illustrative plates of plasmid pK18mobsacBpck1_1
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
BamHI: the cleavage site of Restriction Enzyme BamHI
LysC:lysC FBRAllelotrope, lysC T311I
Pck ': 5 ' terminal fragment of pck gene
3 ' terminal fragment of ' pck:pck gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 4: the collection of illustrative plates of plasmid pK18mobsacBgluB2_1.
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
The cleavage site of SalI Restriction Enzyme SalI
The cleavage site of EcoRI Restriction Enzyme EcoRI
BamHI: the cleavage site of Restriction Enzyme BamHI
The ddh:ddh gene
GluA gluA gene
GluB ': 5 ' terminal fragment of gluB gene
3 ' terminal fragment of ' gluB:gluB gene
GluC gluC gene
GluD ': 5 ' terminal fragment of gluD gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 5: the collection of illustrative plates of plasmid pK18mobsacBaecD2_1.
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
The cleavage site of EooRI Restriction Enzyme EooRI
SalI: the cleavage site of Restriction Enzyme SalI
The dapA:dapA gene
AecD ': 5 ' terminal fragment of aecD gene
3 ' terminal fragment of ' aecD:aecD gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 6: the collection of illustrative plates of plasmid pK18mobsacBpck1_3.
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
Pyc:pyc allelotrope, pyc P458S
Pck ': 5 ' terminal fragment of pck gene
3 ' terminal fragment of ' pck:pck gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 7: the collection of illustrative plates of plasmid pK18mobsacBnc ∷ lysC
Employed abbreviation and mark have following implication:
KanR: kalamycin resistance gene
BamHI: the cleavage site of Restriction Enzyme BamHI
The cleavage site of HindIII Restriction Enzyme HindIII
The cleavage site of XbaI Restriction Enzyme XbaI
LysC:lysC FBRAllelotrope, lysC T311I
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 8: the collection of illustrative plates of plasmid pK18mobsacBddh ∷ ilvD
Employed abbreviation and mark have following implication:
Kan: kalamycin resistance gene
XbaI: the cleavage site of Restriction Enzyme XbaI
NheI: the cleavage site of Restriction Enzyme NheI
5 ' terminal fragment of ddh int ' ddh gene
3 ' terminal fragment of ' ddh int ddh gene
IlvD ilvD gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 9: the collection of illustrative plates of plasmid pK18mobsacBnc ∷ trp
Employed abbreviation and mark have following implication:
KmR: kalamycin resistance gene
BamHI: the cleavage site of Restriction Enzyme BamHI
HindIII: the cleavage site of property enzyme HindIII
5 ' the terminal fragment in nc ' ncode1 zone
3 ' the terminal fragment in ' nc ncode1 zone
TrpL trpL gene
TrpE trpE gene
TrpG trpG gene
TrpD trpD gene
TrpC trpC gene
TrpB trpB gene
TrpA trpA gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
Accompanying drawing 9: the collection of illustrative plates of plasmid pK18mobsacBph1 ∷ trp
Employed abbreviation and mark have following implication:
KmR: kalamycin resistance gene
BamHI: the cleavage site of Restriction Enzyme BamHI
EcoRI: the cleavage site of Restriction Enzyme EcoRI
SalI: the cleavage site of Restriction Enzyme SalI
5 ' terminal fragment of phage ' phage zone phal
3 ' terminal fragment of ' phage phage zone phal
TrpL trpL gene
TrpE trpE gene
TrpG trpG gene
TrpD trpD gene
TrpC trpC gene
TrpB trpB gene
TrpA trpA gene
The sacB:sacB gene
RP4mob: the mob zone that has transfer replication initial point (oriT)
OriV: replication origin V
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issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1 Indicate the date of original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit ordate of the transfer).
2 In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viabillty test.
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test were negative.Form DSMZ-BP/9(sole page)0196
Be used for the patented procedure purpose
International recognition aspect the microbial preservation
The special treaty in Budapest
INTERNATIONAL FORM
Degussa AG
Kantstr.2
33790Halle(Westf.) RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issred pursuant to Rule 7.1 by the
INTERNATJONAL DEPOSITARY AUTHORITY
identified at the bottorm of this page
1Where Rule 6.4(d)applies，such date is the date on which the status of international depositary authority was acquired.Form DSMZ-BP/4(sole page)12/2001
Be used for the patented procedure purpose
International recognition aspect the microbial preservation
The special treaty in Budapest
Figure A20048000352901201
INTERNATIONAL FORM
Degussa AG
Kantstr. 2
33790 Halle(Westf.)
VIABILTTY STATEMENT
issred pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
Figure A20048000352901202
1 Indicate the date of original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or dateof the transfer).
2 In the cases referred to in Rulc 10.2(e)(ii)and(iii)，refer to the most recent viability test.
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test were negative.Form DSMZ-BP/9(sole page)12/2001
Sequence table
<110>Degussa AG
＜120〉bacterium and with the method for described bacterium I production compound
<130>010301 BT
<160>50
<170>PatentIn version 3.1
<210>1
<211>1263
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>CDS
<222>(1)..(1263)
＜223〉lysC wild type gene
<400>1
gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg 48
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct 96
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
aag atc tgc att gtt get ggt ttc cag ggt gtt aat aaa gaa acc cgc 432
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
ctg gaa aag ctc age ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
gac ggc acc acc gac atc acc ttc acc tgc cct cgt tcc gac ggc cgc 960
Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
gca ggc acc gga cgc 1263
Ala Gly Thr Gly Arg
420
<210>2
<211>421
<212>PRT
＜213〉Corynebacterium glutamicum
<400>2
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
2l0 215 220
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
Ala Gly Thr Gly Arg
420
<210>3
<211>1263
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>CDS
<222>(1)..(1263)
<223>lysC-fbr allele lysC T311I
<400>3
gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg 48
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct 96
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288
Val Ala Met Ala Ile Glu Ser Leu Glv Ala Glu Ala Gln Ser Phe Thr
85 90 95
ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
aag atc tgc att gtt gct ggt ttc cag ggt gtt aat aaa gaa acc cgc 432
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624
Leu Glu Lys Leu Ser Phe Glu Glu MetLeu Glu Leu Ala Ala Val Gly
195 200 205
tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
gac ggc acc acc gac atc atc ttc acc tgc cct cgt tcc gac ggc cgc 960
Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
gca ggc acc gga cgc 1263
Ala Gly Thr Gly Arg
420
<210>4
<211>421
<212>PRT
＜213〉Corynebacterium glutamicum
<400>4
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys G1u I1e Tyr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
Ala Gly Thr Gly Arg
420
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer lysC1beg
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer lysC1beg
<400>5
taggatcctc cggtgtctga ccacggtg 28
<210>6
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer lysC2end
<220>
<221>misc_feature
<222>(1)..(29)
＜223〉primer lysC2end
<400>6
acggatccgc tgggaaattg cgctcttcc 29
<210>7
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer gluBgl1
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer gluBgl1
<400>7
taagatctgt gttggacgtc atggcaag 28
<210>8
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer gluBgl2
<400>8
acagatcttg aagccaagta cggccaag 28
<210>9
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pck_beg
<220>
<221>misc_feature
<222>(1)..(27)
＜223〉primer pck_anf
<220>
<221>misc_feature
<222>(1)..(27)
＜223〉primer pck_beg
<400>9
taagatctgc cggcatgact tcagttt 27
<210>10
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pck_end
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer pck_end
<400>10
acagatctgg tgggagcctt tcttgttatt 30
<210>11
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer aecD_beg
<400>11
gaacttacgc caagctgttc 20
<210>12
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer aecD_end
<400>12
agcaccacaa tcaacgtgag 20
<210>13
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer gluA_beg
<400>13
cacggttgct cattgtatcc 20
<210>14
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer gluD_end
<400>14
cgaggcgaat cagacttctt 20
<210>15
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh_beg
<400>15
ctgaatcaaa ggcggacatg 20
<210>16
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh_end
<400>16
tcgagctaaa ttagacgtcg 20
<210>17
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer dapA_beg
<400>17
cgagccagtg aacatgcaga 20
<210>18
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer dapA_end
<400>18
cttgagcacc ttgcgcagca 20
<210>19
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pyc_beg
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer pyc_anf
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer pyc_beg
<400>19
tcacgcgtct tgaagtcgtg caggtcag 28
<210>20
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pyc_end
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer pyc_end
<400>20
tcacgcgtcg cctcctccat gaggaaga 28
<210>21
<211>39
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(39)
＜223〉primer P458S-1
<400>21
ggattcattg ccgatcactc gcacctcctt caggctcca 39
<210>22
<211>39
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(39)
＜223〉primer P458S-2
<400>22
gtggaggaag tccgaggtcg agtgatcggc aatgaatcc 39
<210>23
<211>1310
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(1310)
＜223〉intergenic region ncode1
<400>23
aagctctatt gtcccctacg tgctcgtttc tggcctttta gtaagcacca ggaataagcg 60
ccgatgaaga cacaatcata ccgacaatta atcgtgccga tatgagctct taaaagacag 120
cataaaacga gtttttcaaa agcctattaa gtgtcaatta cgacgtgcat taatagatac 180
tcaatcacct taaattgttg acacactcca ctaaaacagg tctattaaaa gacaattgaa 240
ttacgcccta gtagtacttg tttcaggcca ccacttagaa ggcttttaag tatccactat 300
gtatcaatta tctagaacct ttagtgactt tgaaacggca gtactctatt ggctcttaat 360
ggtcaattac ataacaatta tattgagcct ttgaaacaac tcactctgct gcatattaaa 420
aggtcgatta actaacgatt gaattgatcc ttaaaaagcc tttatctatc gcattatgaa 480
taaatattta atcgaccttt aatagtgacc taaaagcctt ttaaaagcca acgcattcag 540
tgacttttaa aaggctatta agtgtcaatt gaattgcctt gttactggct tttaaaaggc 600
tattaaagaa cactccccta ttgtctttta atcgtcactt aatcgacctc taaaaggtaa 660
ctaattgact cttgagtgac acatatttaa ttgaccttta agtaacgatt ataaggcaat 720
taatgtgacc aaataaagac acgtaactga ctaatcttta tctgactatt acaaggcttt 780
aaaagagcac ttatgtgtcg attaagtgtc tacgcaataa ctgtgcttta agaggcttta 840
aaaactacaa ttgaatcgac cactaatcgt tacttaaatg actattaaca aaagtcactt 900
ttagagcacc gcaaaagcct tttaatggtc acgcaataag cctttaagta acaattaaat 960
aagtggcttt aaaatcacta ctgcagcacg attgaaaggt aattagcggt cgattaagtg 1020
tcaattaatt aatagtgatt caaaatctca ttaaagcgca attcaattga cagctaataa 1080
gcccttaagt aacaaatact ttatccgtac tttaagggca cgctaaaagc cttttaatcg 1140
acatctaatt gtcataacgc ttcgatgcgc ccatgggaat acacttagcg gtcgattaaa 1200
tggatactaa gtagcaatta gccagagtcg cgtgacagag ttgtggcgca cagtagcaca 1260
tcacccctac ccccgtgcat ctcttaatta gcactaaaac aacatttatc 1310
<210>24
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer ncode 1
<400>24
gaagatctaa gctctattgt cccctacg 28
<210>25
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(1)..(25)
＜223〉primer ncode 2
<400>25
gatcctttta aaagccagta acaag 25
<210>26
<211>47
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(1)..(47)
＜223〉primer ncode 3
<400>26
cttgttactg gcttttaaaa ggatcctatt aaagaacact cccctat 47
<210>27
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer ncode 4
<400>27
gaagatctcg actctggcta attgctac 28
<210>28
<211>1249
<212>DNA
＜213〉artificial sequence
<220>
<223>PCR-Produkt
<220>
<221>misc_feature
<222>(1)..(1249)
＜223〉the intergenic region ncode1 after the PCR
<400>28
gaagatctaa gctctattgt cccctacgtg ctcgtttctg gccttttagt aagcaccagg 60
aataagcgcc gatgaagaca caatcatacc gacaattaat cgtgccgata tgagctctta 120
aaagacagca taaaacgagt ttttcaaaag cctattaagt gtcaattacg acgtgcatta 180
atagatactc aatcacctta aattgttgac acactccact aaaacaggtc tattaaaaga 240
caattgaatt acgccctagt agtacttgtt tcaggccacc acttagaagg cttttaagta 300
tccactatgt atcaattatc tagaaccttt agtgactttg aaacggcagt actctattgg 360
ctcttaatgg tcaattacat aacaattata ttgagccttt gaaacaactc actctgctgc 420
atattaaaag gtcgattaac taacgattga attgatcctt aaaaagcctt tatctatcgc 480
attatgaata aatatttaat cgacctttaa tagtgaccta aaagcctttt aaaagccaac 540
gcattcagtg acttttaaaa ggctattaag tgtcaattga attgccttgt tactggcttt 600
taaaaggatc ctattaaaga acactcccct attgtctttt aatcgtcact taatcgacct 660
ctaaaaggta actaattgac tcttgagtga cacatattta attgaccttt aagtaacgat 720
tataaggcaa ttaatgtgac caaataaaga cacgtaactg actaatcttt atctgactat 780
tacaaggctt taaaagagca cttatgtgtc gattaagtgt ctacgcaata actgtgcttt 840
aagaggcttt aaaaactaca attgaatcga ccactaatcg ttacttaaat gactattaac 900
aaaagtcact tttagagcac cgcaaaagcc ttttaatggt cacgcaataa gcctttaagt 960
aacaattaaa taagtggctt taaaatcact actgcagcac gattgaaagg taattagcgg 1020
tcgattaagt gtcaattaat taatagtgat tcaaaatctc attaaagcgc aattcaattg 1080
acagctaata agcccttaag taacaaatac tttatccgta ctttaagggc acgctaaaag 1140
ccttttaatc gacatctaat tgtcataacg cttcgatgcg cccatgggaa tacacttagc 1200
ggtcgattaa atggatacta agtagcaatt agccagagtc gagatctag 1249
<210>29
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer 3371V
<400>29
tatcatgcgg tgagctgtga 20
<210>30
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer 3372N
<400>30
taggggtgat gtgctactgt 20
<210>31
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer ilvD-A1
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer ilvD-A1
<400>31
atcgtctcta gacttggagc gccaaaaggc ac 32
<210>32
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer ilvD-E1
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer ilvD-E1
<400>32
gtcatctcta gatcgaggca gagttcgctg gt 32
<210>33
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer ddh_del1
<220>
<221>misc_feature
<222>(1)..(33)
＜223〉primer ddh_del1
<400>33
atcgtcgcta gcccaacgaa tctccacgag acg 33
<210>34
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh_del2
<400>34
gacatccgca ccatgcctga 20
<210>35
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer ddh_del3
<220>
<221>misc_feature
<222>(1)..(44)
＜223〉primer ddh_del3
<400>35
tcaggcatgg tgcggatgtc tagagtcggc aaccacgaag catt 44
<210>36
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer ddh_del4
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer ddh_del4
<400>36
atgctcgcta gcatgaccaa catccgcgta gc 32
<210>37
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh-int1
<400>37
attcttccgc gaccgcgata 20
<210>38
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh_A1
<400>38
ccggataaac caccatgaac 20
<210>39
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer trp-A1
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer trp-A1
<400>39
gtacggatcc caaggacagc tcgtgtagac 30
<210>40
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer trp-E1
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer trp-E1
<400>40
agctggatcc caccggctcg aagctaagat 30
<210>41
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ncode1-A1
<400>41
actatcatgc ggtgagctgt 20
<210>42
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ncode1-E1
<400>42
ggeagctctg tagccatatt 20
<210>43
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer trp-test
<400>43
ccaaccacga tgagaaggac 20
<210>44
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pha1-int1
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer pha1-int1
<400>44
agtcgtcgac gctatcaacg cttcgatcac 30
<210>45
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer phal-int2
<400>45
ctgtctcgta cggatgacag 20
<210>46
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pha1-int3
<220>
<221>misc_feature
<222>(1)..(45)
＜223〉primer pha1-int3
<400>46
ctgtcatccg tacgagacag ggatccatac cttgccagtc aaatc 45
<210>47
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer pha1-int4
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer pha1-int4
<400>47
gtcagaattc gcagcatgta acggataggt 30
<210>48
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer pha1-test1
<400>48
gcgctccatc actagagttc 20
<210>49
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer pha1-test2
<400>49
ggtcagagtg agcacctaag 20
<210>50
<211>20
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer pha1-test3
<400>50
ctgcgagctt cattcgatcc 20

Claims (36)

1. coryneform bacterium, it is used for the protein of synthetic compound or the open reading-frame (ORF) of RNA (ORF), gene or the allelotrope except one or more coding that contains a copy at natural site (seat),
A) also be integrated into described open reading-frame (ORF) (ORF), gene or the allelotrope that chromosomal site contains one or more copy, thus
B) do not contain in microorganism can/make it possible to duplicate or the nucleotide sequence of swivel base and
C) do not contain in described site the Nucleotide of giving antibiotics resistance and
D) described site is irrelevant with the vital open reading-frame (ORF) of production (ORF), gene or allelotrope for the growth of described bacterium and purpose compound,
E) at least one described site is selected from the DNA of intergenic region, prophage and defective phage or phage assembly or the described prophage of encoding, defective phage or phage assembly.
2. according to the coryneform bacterium of claim 1, wherein intergenic region is identical with the polynucleotide at least 70% that are selected from following table Reference Accession number Sequence start position The EOS position EP1108790 AX120085 192176 194501 EP1108790 AX127145 235840 237311 EP1108790 AX127145 236096 237311 EP1108790 AX127148 322628 330877 EP1108790 AX127148 334045 336467 EP1108790 AX127148 289565 291841 EP1108790 AX127149 154823 161111 EP1108790 AX127149 190088 193497 EP1108790 AX127149 27398 28707 EP1108790 AX127149 61478 62944 EP1108790 AX127149 116234 117561 EP1108790 AX127149 140847 144605 EP1108790 AX127150 113274 114324 EP1108790 AX127152 244281 246403
。
3. according to the coryneform bacterium of claim 1, it is identical with the polynucleotide at least 70% of selecting following table wherein to be included in intrachromosomal prophage, defective phage or phage assembly Reference Accession number Sequence start position The EOS position EP1108790 AX127149 50474 51049 EP1108790 AX127149 67886 68587 EP1108790 AX127151 72893 73480 EP1108790 AX127149 88231 89445 EP1108790 AX127148 139781 140155 EP1108790 AX127148 140546 141001 EP1108790 AX127149 194608 195294 EP1108790 AX127147 200185 200940 EP1108790 AX127147 208157 208450 EP1108790 AX127149 269616 269948 EP1108790 AX127148 336468 338324 EP1108790 AX127148 342235 342681 EP1108790 AX127148 343518 345356 EP1108790 AX127148 345872 346207
。
4. according to the coryneform bacterium of claim 1, wherein said coryneform bacterium belongs to Corynebacterium.
5. according to the coryneform bacterium of the Corynebacterium of claim 2, they belong to the Corynebacterium glutamicum species.
6. according to the coryneform bacterium of claim 1, wherein said compound is selected from L-amino acid, VITAMIN, nucleosides and Nucleotide.
7. according to the coryneform bacterium of claim 1, wherein compound is that one or more is selected from the arginic L-amino acid of L-aspartic acid, altheine, L-Threonine, L-Serine, L-L-glutamic acid, L-glutaminate, glycine, L-L-Ala, L-halfcystine, L-Xie Ansuan, L-methionine(Met), L-Isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-Histidine, L-Methionin, L-tryptophane, L-proline(Pro) and L-.
8. according to the coryneform bacterium of claim 6, wherein L-amino acid is L-Methionin.
9. coryneform bacterium according to Claim 8, wherein coding proteinic one or more the described open reading-frame (ORF) (ORF), gene or the allelotrope that are used to produce Methionin is selected from accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC FBR, lysE, msiK, opcA, oxyR, ppc, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T.
10. coryneform bacterium according to Claim 8, wherein coding proteinic one or more the described open reading-frame (ORF), gene or the allelotrope that are used to produce Methionin is selected from dapA, ddh, lysC FBRWith pyc P458S.
11. coryneform bacterium according to Claim 8 wherein contains the lysC of the E.C. 2.7.2.4. of encoder feedback resistance form FBRAllelotrope.
12. the coryneform bacterium according to claim 11 wherein contains by lysC FBRAllelotrope coding, has the feedback resistance E.C. 2.7.2.4. that comprises one or more aminoacid replacement that is selected from A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F according to the aminoacid sequence of SEQ ID NO:2.
13. according to the coryneform bacterium of claim 11, wherein by lysC FBRThe feedback resistance E.C. 2.7.2.4. of allelotrope coding comprises the aminoacid sequence according to SEQ ID NO:4.
14. according to the coryneform bacterium of claim 11, wherein lysC FBRThe allelotrope coding region comprises the nucleotide sequence of SEQ ID NO:3.
15. coryneform bacterium according to Claim 8, wherein said gene or the allelic the 3rd or the 4th copy are incorporated into the site that is selected from aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi and poxB.
16. according to the coryneform bacterium of claim 15, wherein the site is the aecD gene locus.
17. according to the coryneform bacterium of the production L-Methionin of claim 151, wherein the site is the gluB gene locus.
18. according to the coryneform bacterium of the production L-Methionin of claim 15, wherein the site is the pck gene locus.
19. prepare the method for compound, be included in that the coryneform bacterium to claim 1 to 18 ferments in the appropriate media.
20. prepare the method for compound by the fermentation coryneform bacterium, wherein carry out following steps:
A) fermentation coryneform bacterium, this coryneform bacterium is used for open reading-frame (ORF) (ORF), gene or the allelotrope of the protein of synthetic described compound or RNA except there is one or more coding of at least one copy at natural site (seat),
B) also contain one or more being integrated into chromosomal site, especially second, described open reading-frame (ORF) (ORF), gene or the allelotrope of the 3rd or the 4th copy alternatively, fermentation is carried out under the condition that described open reading-frame (ORF) (ORF), gene or allelotrope are expressed, thus
C) do not have in microorganism can/make it possible to add build duplicates or the nucleotide sequence of swivel base and
D) do not have the nucleotide sequence of giving its antibiotics resistance in described site, thus
E) at least one described site is selected from the DNA that all are included in intrachromosomal intergenic region, prophage, defective phage or phage assembly or the described prophage of encoding, defective phage or phage assembly,
F) compound in concentrated broth body substratum and/or the bacterial cell,
G) separate this compound, alternatively
H) together with from the composition in fermentation broth and/or the biomass, until＞(greater than) 0 to 100wt%.
21. according to the method for claim 20, wherein intergenic region is selected from the form of claim 2.
22. according to the method for claim 20, wherein said prophage, defective phage or phage assembly are selected from the form of claim 3.
23. method according to claim 20, wherein used such coryneform bacterium, the intracellular reactive of the respective egg white matter of being encoded by described open reading-frame (ORF), gene or allelotrope in these bacteriums is enhanced, and these proteinic nucleotide sequences of especially encoding are crossed expression.
24. according to the method for claim 20 to 29, wherein said bacterium belongs to Corynebacterium.
25. according to the method for claim 24, wherein said bacterium belongs to the Corynebacterium glutamicum species.
26. according to the method for claim 20 to 25, wherein compound is selected from L-amino acid, VITAMIN, nucleosides and Nucleotide.
27. according to the method for claim 27, wherein compound is that one or more is selected from the arginic L-amino acid of L-aspartic acid, altheine, L-Threonine, L-Serine, L-L-glutamic acid, L-glutaminate, glycine, L-L-Ala, L-halfcystine, L-Xie Ansuan, L-methionine(Met), L-Isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-Histidine, L-Methionin, L-tryptophane, L-proline(Pro) and L-.
28. according to the method for claim 26, wherein compound is a L-Methionin.
29. the method for preparing L-Methionin according to claim 20 to 26, wherein used such coryneform bacterium, one or more open reading-frame (ORF), gene or allelotrope are selected from accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC in these bacteriums FBR, lysE, msiK, opcA, oxyR, ppc, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pycP458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T.
30. according to the method for preparing L-Methionin of claim 20 to 26, wherein ferment one or more open reading-frame (ORF), gene or allelotrope are selected from gene or allelotrope dapA, ddh, lysC FBRCoryneform bacterium with pyc P458S.
31. according to the method for preparing L-Methionin of claim 30, wherein allelotrope is the lysC of encoder feedback resistance E.C. 2.7.2.4. FBRAllelotrope.
32. according to the method for preparing L-Methionin of claim 31, wherein by lysC FBRThe feedback resistance E.C. 2.7.2.4. of allelotrope coding comprises the aminoacid sequence according to SEQ ID NO:2 that contains one or more aminoacid replacement that is selected from A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F.
33. according to the method for preparing L-Methionin of claim 31, wherein by lysC FBRThe feedback resistance E.C. 2.7.2.4. of allelotrope coding comprises the aminoacid sequence according to SEQ ID NO:4.
34. according to the method for preparing L-Methionin of claim 31, wherein lysC FBRThe allelotrope coding region comprises the nucleotide sequence of SEQ ID NO:3.
35. ferment according to the method for preparing L-Methionin of claim 20 to 26, the coryneform bacterium that wherein the 3rd or the 4th site is selected from aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menB, mqo, pck, pgi and poxB.
36. produce the method for coryneform bacterium, comprising:
A) preferably from coryneform bacterium separating at least one coding be used for the protein of production compound or purpose ORF, gene or the allelic nucleotide sequence of RNA, comprise alternatively and expressing and/or adjustment signal,
B) for described ORF, gene or allelic 5 ' and 3 ' end the nucleotide sequence of target site is provided,
C) wherein said site is selected from intergenic region, prophage, defective phage or phage assembly,
D) purpose ORF, gene or the allelic nucleotide sequence that preferably will have a target site nucleotide sequence is integrated into and do not duplicate in coryneform bacterium or only carry out in the limited suitable carrier that duplicates,
E) with b) or described nucleotides sequence column jump c) is advanced in the coryneform bacterium and
F) be separated in target site and integrated coryneform bacterium according to a) nucleotide sequence.
CNA2004800035293A 2003-02-05 2004-01-29 Bacteria and process for producing chemical compounds by said bacteria Pending CN1748031A (en)

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